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.

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.

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.

Persister Cells Form in the Plant Pathogen Xanthomonas citri subsp. citri under Different Stress Conditions.

Citrus canker disease, caused by the bacterium Xanthomonas citri subsp. citri is a constant threat to citrus-producing areas. Since it has no cure, agricultural practices to restrain its dissemination are essential to reduce the economic damage. Hence, increased knowledge of the basic aspects of X. citri biology could lead to more efficient management practices that can eliminate dormant bacteria in the field. The dormant cells, also referred to as persisters, are phenotypic variants with lowered metabolism, which in turn leads to tolerance to antimicrobials and undermines existing control approaches. We show here that X. citri forms persisters, identifying triggers for this phenotype, including antibiotics, high temperature, and metals (copper and zinc), which increase persistence rates by 10-100 times. The antioxidant N-acetylcysteine reduced copper and zinc-induced persisters, but not those induced by tetracycline, indicating that oxidative stress may be an important inducer of X. citri persistence. In addition, we found that metabolism-independent drugs like cisplatin and mitomycin C are able to eliminate X. citri persistent cells, as well as copper, at high concentrations. Specific amino acids like proline and isoleucine interfered with the physiological balance of the dormancy in X. citri, stimulating or preventing persister resuscitation. Taken together, we discover chemicals that can induce, wake, and kill X. citri persister cells; these results provide insights that should be considered for more efficient integrated control management in the field.

Functionalized microchannels as xylem-mimicking environment: Quantifying X. fastidiosa cell adhesion.

Microchannels can be used to simulate xylem vessels and investigate phytopathogen colonization under controlled conditions. In this work, we explore surface functionalization strategies for polydimethylsiloxane and glass microchannels to study microenvironment colonization by Xylella fastidiosa subsp. pauca cells. We closely monitored cell initial adhesion, growth, and motility inside microfluidic channels as a function of chemical environments that mimic those found in xylem vessels. Carboxymethylcellulose (CMC), a synthetic cellulose, and an adhesin that is overexpressed during early stages of X. fastidiosa biofilm formation, XadA1 protein, were immobilized on the device's internal surfaces. This latter protocol increased bacterial density as compared with CMC. We quantitatively evaluated the different X. fastidiosa attachment affinities to each type of microchannel surface using a mathematical model and experimental observations acquired under constant flow of culture medium. We thus estimate that bacterial cells present ∼4 and 82% better adhesion rates in CMC- and XadA1-functionalized channels, respectively. Furthermore, variable flow experiments show that bacterial adhesion forces against shear stresses approximately doubled in value for the XadA1-functionalized microchannel as compared with the polydimethylsiloxane and glass pristine channels. These results show the viability of functionalized microchannels to mimic xylem vessels and corroborate the important role of chemical environments, and particularly XadA1 adhesin, for early stages of X. fastidiosa biofilm formation, as well as adhesivity modulation along the pathogen life cycle.

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.

Copper Kills Escherichia coli Persister Cells.

Due to their reduced metabolism, persister cells can survive most antimicrobial treatments, which usually rely on corrupting active biochemical pathways. Therefore, molecules that kill bacterial persisters should function in a metabolism-independent manner. Some anti-persister compounds have been found previously, such as the DNA-crosslinkers mitomycin C and cisplatin, but more effective and lower cost alternatives are needed. Copper alloys have been used since ancient times due to their antimicrobial properties, and they are still used in agriculture to control plant bacterial diseases. By stopping transcription with rifampicin and by treating with ampicillin to remove non-persister cells, we created a population that consists solely of Escherichia coli persister cells. Using this population of persister cells, we demonstrate that cupric compounds kill E. coli persister cells. Hence, copper ions may be used in controlling the spread of important bacterial strains that withstand treatment with conventional antimicrobials by forming persister cells.

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.

Stiffness signatures along early stages of Xylella fastidiosa biofilm formation.

The pathogenicity of Xylella fastidiosa is associated with its systematic colonization of the plant xylem, forming bacterial biofilms. Mechanisms of bacterial transport among different xylem vessels, however, are not completely understood yet, but are strongly influenced by the presence of extracellular polymeric substances (EPS), which surrounds the assembly of cells forming the biofilm. In this work, we show quantitative measurements on the elastic properties of the system composed by EPS and bacterial cell. In order to investigate the mechanical properties of this system, force spectroscopy and confocal Raman measurements were carried out during Xylella fastidiosa subsp. pauca initial stages of adhesion and cluster formation. We show that stiffness progressively decreases with increasing culture growth time, from two to five days. For early adhesion samples, stiffness values are quite different at the bacterial polar and body regions. Lower stiffness values at the cell pole suggest a flexible mechanical response at this region, associated with first cell adhesion to a surface. These results correlate very well with our observations of cell motion within microchannels, under conditions simulating xylem flow. Both the oscillatory movement of vertically attached single cells, as well as the transport of cell clusters within the biofilm matrix can be explained by the presence of softer materials at the cell pole and EPS matrix. Our results may thus add to a more detailed understanding of mechanisms used by cells to migrate among vessels in plant xylem.

PAMPs, PRRs, effectors and R-genes associated with citrus-pathogen interactions.

BACKGROUND: Recent application of molecular-based technologies has considerably advanced our understanding of complex processes in plant-pathogen interactions and their key components such as PAMPs, PRRs, effectors and R-genes. To develop novel control strategies for disease prevention in citrus, it is essential to expand and consolidate our knowledge of the molecular interaction of citrus plants with their pathogens. SCOPE: This review provides an overview of our understanding of citrus plant immunity, focusing on the molecular mechanisms involved in the interactions with viruses, bacteria, fungi, oomycetes and vectors related to the following diseases: tristeza, psorosis, citrus variegated chlorosis, citrus canker, huanglongbing, brown spot, post-bloom, anthracnose, gummosis and citrus root rot.

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.

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.

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.

Nanofilms of hyaluronan/chitosan assembled layer-by-layer: An antibacterial surface for Xylella fastidiosa.

In this work, nanofilms of hyaluronan/chitosan (HA/CHI) assembled layer by layer were synthesized; their application as a potential antimicrobial material was demonstrated for the phytopathogen Xylella fastidiosa, a gram-negative bacterium, here used as a model. For the synthesis, the influence of pH and ionic strength of these natural polymer stem-solutions on final characteristics of the HA/CHI nanofilms was studied in detail. The antibacterial effect was evaluated using widefield fluorescence microscopy. These results were correlated with the chemical properties of the nanofilms, studied by FTIR and Raman spectroscopy, as well as with their morphology and surface properties characterized using SEM and AFM. The present findings can be extended to design and optimize HA/CHI nanofilms with enhanced antimicrobial behavior for other type of phytopathogenic gram-negative bacteria species, such as Xanthomonas citri, Xanthomas campestri and Ralstonia solanacearum.

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.

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.