Resolving the metabolism of monolignols and other lignin-related aromatic compounds in Xanthomonas citri.

Lignin, a major plant cell wall component, has an important role in plant-defense mechanisms against pathogens and is a promising renewable carbon source to produce bio-based chemicals. However, our understanding of microbial metabolism is incomplete regarding certain lignin-related compounds like p-coumaryl and sinapyl alcohols. Here, we reveal peripheral pathways for the catabolism of the three main lignin precursors (p-coumaryl, coniferyl, and sinapyl alcohols) in the plant pathogen Xanthomonas citri. Our study demonstrates all the necessary enzymatic steps for funneling these monolignols into the tricarboxylic acid cycle, concurrently uncovering aryl aldehyde reductases that likely protect the pathogen from aldehydes toxicity. It also shows that lignin-related aromatic compounds activate transcriptional responses related to chemotaxis and flagellar-dependent motility, which might play an important role during plant infection. Together our findings provide foundational knowledge to support biotechnological advances for both plant diseases treatments and conversion of lignin-derived compounds into bio-based chemicals.

VirB11, a traffic ATPase, mediated flagella assembly and type IV pilus morphogenesis to control the motility and virulence of Xanthomonas albilineans.

Xanthomonas albilineans (Xal) is a gram-negative bacterial pathogen responsible for developing sugarcane leaf scald disease, which engenders significant economic losses within the sugarcane industry. In the current study, homologous recombination exchange was carried out to induce mutations within the virB/D4-like type IV secretion system (T4SS) genes of Xal. The results revealed that the virB11-deletion mutant (ΔvirB11) exhibited a loss in swimming and twitching motility. Application of transmission electron microscopy analysis further demonstrated that the ΔvirB11 failed to develop flagella formation and type IV pilus morphology and exhibited reduced swarming behaviour and virulence. However, these alterations had no discernible impact on bacterial growth. Comparative transcriptome analysis between the wild-type Xal JG43 and the deletion-mutant ΔvirB11 revealed 123 differentially expressed genes (DEGs), of which 28 and 10 DEGs were notably associated with flagellar assembly and chemotaxis, respectively. In light of these findings, we postulate that virB11 plays an indispensable role in regulating the processes related to motility and chemotaxis in Xal.

Taxonogenomic analysis of the Xanthomonas translucens complex leads to the descriptions of Xanthomonas cerealis sp. nov. and Xanthomonas graminis sp. nov.

The pathovar-based taxonomy of the Xanthomonas translucens group is very confusing due to an overlap of plant host ranges and level of host specificity. Here, whole-genome sequence-based parameters (digital DNA-DNA hybridization and blast-based average nucleotide identity), phylogenomic, biochemical and phenotypical data were used to taxonomically analyse the 11 known pathovars of the X. translucens complex. This polyphasic approach taxonomically assigned the 11 pathovars of X. translucens complex into three distinct species, two of which are new: X. translucens, X. cerealis sp. nov. and X. graminis sp. nov. X. translucens consists of three pathovars: pv. translucens (=pv. hordei), pv. pistaciae strain A ICMP 16316PT and pv. undulosa (=pv. secalis). X. cerealis sp. nov. encompasses the pv. cerealis strain LMG 679PT and pv. pistaciae strain B ICMP 16317PT with genome similarity of 92.7% (dDDH) and 99.0% (ANIb) suggesting taxonomically similar genotypes. The other new species, X. graminis sp. nov., consists of the remaining five designated pathovars (pv. graminis, pv. arrhenatheri, pv. poae, pv. phleipratensis and pv. phlei) with highly variable dDDH and ANIb values ranging from 74.5 to 93.0% and from 96.7 to 99.2%, respectively, an indication of a very divergent taxonomic group. Only strains of pvs. phlei and phleipratensis showed the highest genomic similarities of 93.0% (dDDH) and 99.2% (ANIb), suggesting synonymic pathovars as both infect the same plant hosts. The dDDH and ANI data were corroborated by phylogenomics clustering. The fatty acid contents were similar but the type strain of X. graminis sp. nov. exhibited 20% less C15 : 0 iso and 40% more C17 : 0 iso fatty acids than the other species. Based on phenotypic, biochemical and whole-genome sequence data, we propose two new species, Xanthomonas cerealis sp. nov. and Xanthomonas graminis sp. nov. with type strains LMG 679T (=NCPPB 1944T) and LMG 726T (=NCPPB 2700T), respectively.

Exploring Fatty Acid ß-Oxidation Pathways in Bacteria: From General Mechanisms to DSF Signaling and Pathogenicity in Xanthomonas.

Fatty acids (FAs) participate in extensive physiological activities such as energy metabolism, transcriptional control, and cell signaling. In bacteria, FAs are degraded and utilized through various metabolic pathways, including ß-oxidation. Over the past ten years, significant progress has been made in studying FA oxidation in bacteria, particularly in E. coli, where the processes and roles of FA ß-oxidation have been comprehensively elucidated. Here, we provide an update on the new research achievements in FAs ß-oxidation in bacteria. Using Xanthomonas as an example, we introduce the oxidation process and regulation mechanism of the DSF-family quorum sensing signal. Based on current findings, we propose the specific enzymes required for ß-oxidation of several specific FAs. Finally, we discuss the future outlook on scientific issues that remain to be addressed. This paper supplies theoretical guidance for further study of the FA ß-oxidation pathway with particular emphasis on its connection to the pathogenicity mechanisms of bacteria.

Comparative genomics-based insights into Xanthomonas indica, a non-pathogenic species of healthy rice microbiome with bioprotection function.

Xanthomonas species are major pathogens of plants and have been studied extensively. There is increasing recognition of the importance of non-pathogenic species within the same genus. With this came the need to understand the genomic and functional diversity of non-pathogenic Xanthomonas (NPX) at the species and strain level. This study reports isolation and investigation into the genomic diversity and variation in NPX isolates, chiefly Xanthomonas indica, a newly discovered NPX species from rice. The study establishes the relationship of X. indica strains within clade I of Xanthomonads with another NPX species, X. sontii, also associated with rice seeds. Identification of highly diverse strains, open-pan genome, and systematic hyper-variation at the lipopolysaccharide biosynthetic locus when compared to pathogenic Xanthomonas indicates the acquisition of new functions for adaptation. Furthermore, comparative genomics studies established the absence of major virulence genes such as type III secretion system and effectors, which are present in the pathogens, and the presence of a known bacterial-killing type IV secretion system (X-T4SS). The diverse non-pathogenic strains of X. indica and X. sontii were found to protect rice from bacterial leaf blight pathogen, X. oryzae pv. oryzae (Xoo). The absence of phenotype of an X-T4SS mutant suggests redundancy in the genetic basis of the mechanisms involved in the bioprotection function, which may include multiple genetic loci, such as putative bacteriocin-encoding gene clusters and involvement of other factors such as nutrient and niche competition apart from induction of innate immunity through shared microbial-associated molecular patterns. The rice-NPX community and its pathogenic counterpart can be a promising model for understanding plant-microbe-microbiome interaction studies.IMPORTANCEThe Xanthomonas group of bacteria is known for its characteristic lifestyle as a phytopathogen. However, the discovery of non-pathogenic Xanthomonas (NPX) species is a major shift in understanding this group of bacteria. Multi-strain, in-depth genomic, evolutionary and functional studies on each of these NPX species are still lacking. This study on diverse non-pathogenic strains provides novel insights into genome diversity, dynamics, and evolutionary trends of NPX species from rice microbiome apart from its relationship with other relatives that form a sub-clade. Interestingly, we also uncovered that NPX species protect rice from pathogenic Xanthomonas species. The plant protection property shows their importance as a part of a healthy plant microbiome. Furthermore, finding an open pan-genome and large-scale variation at lipopolysaccharide biosynthetic locus indicates a significant role of the NPX community in host adaptation. The findings and high-quality genomic resources of NPX species and the strains will allow further systematic molecular and host-associated microbial community studies for plant health.

Comparative transcriptomics reveals a highly polymorphic Xanthomonas HrpG virulence regulon.

BACKGROUND: Bacteria of the genus Xanthomonas cause economically significant diseases in various crops. Their virulence is dependent on the translocation of type III effectors (T3Es) into plant cells by the type III secretion system (T3SS), a process regulated by the master response regulator HrpG. Although HrpG has been studied for over two decades, its regulon across diverse Xanthomonas species, particularly beyond type III secretion, remains understudied. RESULTS: In this study, we conducted transcriptome sequencing to explore the HrpG regulons of 17 Xanthomonas strains, encompassing six species and nine pathovars, each exhibiting distinct host and tissue specificities. We employed constitutive expression of plasmid-borne hrpG*, which encodes a constitutively active form of HrpG, to induce the regulon. Our findings reveal substantial inter- and intra-specific diversity in the HrpG* regulons across the strains. Besides 21 genes directly involved in the biosynthesis of the T3SS, the core HrpG* regulon is limited to only five additional genes encoding the transcriptional activator HrpX, the two T3E proteins XopR and XopL, a major facility superfamily (MFS) transporter, and the phosphatase PhoC. Interestingly, genes involved in chemotaxis and genes encoding enzymes with carbohydrate-active and proteolytic activities are variably regulated by HrpG*. CONCLUSIONS: The diversity in the HrpG* regulon suggests that HrpG-dependent virulence in Xanthomonas might be achieved through several distinct strain-specific strategies, potentially reflecting adaptation to diverse ecological niches. These findings enhance our understanding of the complex role of HrpG in regulating various virulence and adaptive pathways, extending beyond T3Es and the T3SS.

Xanthomonas citri subsp. citri type III effector PthA4 directs the dynamical expression of a putative citrus carbohydrate-binding protein gene for canker formation.

Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker, elicits canker symptoms in citrus plants because of the transcriptional activator-like (TAL) effector PthA4, which activates the expression of the citrus susceptibility gene CsLOB1. This study reports the regulation of the putative carbohydrate-binding protein gene Cs9g12620 by PthA4-mediated induction of CsLOB1 during Xcc infection. We found that the transcription of Cs9g12620 was induced by infection with Xcc in a PthA4-dependent manner. Even though it specifically bound to a putative TAL effector-binding element in the Cs9g12620 promoter, PthA4 exerted a suppressive effect on the promoter activity. In contrast, CsLOB1 bound to the Cs9g12620 promoter to activate its expression. The silencing of CsLOB1 significantly reduced the level of expression of Cs9g12620, which demonstrated that Cs9g12620 was directly regulated by CsLOB1. Intriguingly, PhtA4 interacted with CsLOB1 and exerted feedback control that suppressed the induction of expression of Cs9g12620 by CsLOB1. Transient overexpression and gene silencing revealed that Cs9g12620 was required for the optimal development of canker symptoms. These results support the hypothesis that the expression of Cs9g12620 is dynamically directed by PthA4 for canker formation through the PthA4-mediated induction of CsLOB1.

Effect of mutation of phaC on carbon supply, extracellular polysaccharide production, and pathogenicity of Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight in rice. Polyhydroxyalkanoates (PHAs) consitute a diverse group of biopolyesters synthesized by bacteria under nutrient-limited conditions. The phaC gene is important for PHA polymerization. We investigated the effects of phaC gene mutagensis in Xoo strain PXO99A. The phaC gene knock-out mutant exhibited reduced swarming ability relative to that of the wild-type. Under conditions where glucose was the sole sugar source, extracellular polysaccharide (EPS) production by ΔphaC declined by 44.8%. ΔphaC showed weak hypersensitive response (HR) induction in the leaves of non-host Nicotiana tabacum, concomitant with downregulation of hpa1 gene expression. When inoculated in rice leaves by the leaf-clipping method, ΔphaC displayed reduced virulence in terms of lesion length compared with the wild-type strain. The complemented strain showed no significant difference from the wild-type strain, suggesting that the deletion of phaC in Xoo induces significant alterations in various physiological and biological processes. These include bacterial swarming ability, EPS production, transcription of hrp genes, and glucose metabolism. These changes are intricately linked to the energy utilization and virulence of Xoo during plant infection. These findings revealed involvement of phaC in Xoo is in the maintaining carbon metabolism by functioning in the PHA metabolic pathway.

The underground world of plant disease: Rhizosphere dysbiosis reduces above-ground plant resistance to bacterial leaf spot and alters plant transcriptome.

Just as the human gut microbiome is colonized by a variety of microbes, so too is the rhizosphere of plants. An imbalance in this microbial community, known as dysbiosis, can have a negative impact on plant health. This study sought to explore the effect of rhizosphere dysbiosis on the health of tomato plants (Solanum lycopersicum L.), using them and the foliar bacterial spot pathogen Xanthomonas perforans as model organisms. The rhizospheres of 3-week-old tomato plants were treated with either streptomycin or water as a control, and then spray-inoculated with X. perforans after 24 h. Half of the plants that were treated with both streptomycin and X. perforans received soil microbiome transplants from uninfected plant donors 48 h after the streptomycin was applied. The plants treated with streptomycin showed a 26% increase in disease severity compared to those that did not receive the antibiotic. However, the plants that received the soil microbiome transplant exhibited an intermediate level of disease severity. The antibiotic-treated plants demonstrated a reduced abundance of rhizobacterial taxa such as Cyanobacteria from the genus Cylindrospermum. They also showed a down-regulation of genes related to plant primary and secondary metabolism, and an up-regulation of plant defence genes associated with induced systemic resistance. This study highlights the vital role that beneficial rhizosphere microbes play in disease resistance, even against foliar pathogens.

A cyclic di-GMP-binding adaptor protein interacts with a N5-glutamine methyltransferase to regulate the pathogenesis in Xanthomonas citri subsp. citri.

The second messenger cyclic diguanylate monophosphate (c-di-GMP) regulates a wide range of bacterial behaviours through diverse mechanisms and binding receptors. Single-domain PilZ proteins, the most widespread and abundant known c-di-GMP receptors in bacteria, act as trans-acting adaptor proteins that enable c-di-GMP to control signalling pathways with high specificity. This study identifies a single-domain PilZ protein, XAC3402 (renamed N5MapZ), from the phytopathogen Xanthomonas citri subsp. citri (Xcc), which modulates Xcc virulence by directly interacting with the methyltransferase HemK. Through yeast two-hybrid, co-immunoprecipitation and immunofluorescent staining, we demonstrated that N5MapZ and HemK interact directly under both in vitro and in vivo conditions, with the strength of the protein-protein interaction decreasing at high c-di-GMP concentrations. This finding distinguishes N5MapZ from other characterized single-domain PilZ proteins, as it was previously known that c-di-GMP enhances the interaction between those single-domain PilZs and their protein partners. This observation is further supported by the fact that the c-di-GMP binding-defective mutant N5MapZR10A can interact with HemK to inhibit the methylation of the class 1 translation termination release factor PrfA. Additionally, we found that HemK plays an important role in Xcc pathogenesis, as the deletion of hemK leads to extensive phenotypic changes, including reduced virulence in citrus plants, decreased motility, production of extracellular enzymes and stress tolerance. Gene expression analysis has revealed that c-di-GMP and the HemK-mediated pathway regulate the expression of multiple virulence effector proteins, uncovering a novel regulatory mechanism through which c-di-GMP regulates Xcc virulence by mediating PrfA methylation via the single-domain PilZ adaptor protein N5MapZ.

VmsR, a LuxR-Type Regulator, Contributes to Virulence, Cell Motility, Extracellular Polysaccharide Production and Biofilm Formation in Xanthomonas oryzae pv. oryzicola.

LuxR-type regulators play pivotal roles in regulating numerous bacterial processes, including bacterial motility and virulence, thereby exerting a significant influence on bacterial behavior and pathogenicity. Xanthomonas oryzae pv. oryzicola, a rice pathogen, causes bacterial leaf streak. Our research has identified VmsR, which is a response regulator of the two-component system (TCS) that belongs to the LuxR family. These findings of the experiment reveal that VmsR plays a crucial role in regulating pathogenicity, motility, biofilm formation, and the production of extracellular polysaccharides (EPSs) in Xoc GX01. Notably, our study shows that the vmsR mutant exhibits a reduced swimming motility but an enhanced swarming motility. Furthermore, this mutant displays decreased virulence while significantly increasing EPS production and biofilm formation. We have uncovered that VmsR directly interacts with the promoter regions of fliC and fliS, promoting their expression. In contrast, VmsR specifically binds to the promoter of gumB, resulting in its downregulation. These findings indicate that the knockout of vmsR has profound effects on virulence, motility, biofilm formation, and EPS production in Xoc GX01, providing insights into the intricate regulatory network of Xoc.

Xanthomonas oryzae pv. oryzicola effector Tal10a directly activates rice OsHXK5 expression to facilitate pathogenesis.

Bacterial leaf streak (BLS), caused by Xanthomonas oryzae pv. oryzicola (Xoc), is a major bacterial disease in rice. Transcription activator-like effectors (TALEs) from Xanthomonas can induce host susceptibility (S) genes and facilitate infection. However, knowledge of the function of Xoc TALEs in promoting bacterial virulence is limited. In this study, we demonstrated the importance of Tal10a for the full virulence of Xoc. Through computational prediction and gene expression analysis, we identified the hexokinase gene OsHXK5 as a host target of Tal10a. Tal10a directly binds to the gene promoter region and activates the expression of OsHXK5. CRISPR/Cas9-mediated gene editing in the effector binding element (EBE) of OsHXK5 significantly increases rice resistance to Xoc, while OsHXK5 overexpression enhances the susceptibility of rice plants and impairs rice defense responses. Moreover, simultaneous editing of the promoters of OsSULTR3;6 and OsHXK5 confers robust resistance to Xoc in rice. Taken together, our findings highlight the role of Tal10a in targeting OsHXK5 to promote infection and suggest that OsHXK5 represents a potential target for engineering rice resistance to Xoc.

Pyramiding of bacterial blight resistance genes into promising restorer BRRI31R line through marker-assisted backcross breeding and evaluation of agro-morphological and physiochemical characteristics of developed resistant restorer lines.

BRRI31R is one of the Bangladesh's most promising restorer lines due to its abundant pollen producing capacity, strong restoring ability, good combining ability, high outcrossing rate and genetically diverse from cytoplasmic male sterile (CMS) line. But the drawback of this line is that it is highly susceptible to bacterial blight (BB) disease of rice caused by Xanthomonas oryzae pv. oryzae. The present study highlighted the pyramiding of effective BB resistance genes (xa5, xa13 and Xa21) into the background of BRRI31R, through marker-assisted backcrossing (MABC). Backcross progenies were confirmed and advanced based on the foreground selection of target genes. Pyramided lines were used for pathogenicity test against five Bangladeshi Xanthomonas oryzae (BXo) races (BXo93, BXo220, BXo822, BXo826, BXo887) and confirmed the dominant fertility restore genes, Rf3 and Rf4 and further validated against SNP markers for more confirmation of target resistance genes. All pyramided restorer lines consisted of Xa4 (in built), xa5, xa13, Xa21, and Chalk5 with two fertility restorer genes, Rf3, Rf4. and these restorer lines showed intermediate amylose content (<25%). Restorer lines BRRI31R-MASP3 and BRRI31R-MASP4 showed high levels of resistance against five virulent BXo races and SNP genotyping revealed that these lines also contained a blast resistance gene Pita races. Gene pyramided restorer lines, BRRI31R-MASP3 and BRRI31R-MASP4 can directly be used as a male parent for the development of new BB resistant hybrid rice variety or could be used as a replacement of restorer line of BRRI hybrid dhan5 and 7 to enhance the quality of hybrid seeds as well as rice production in Bangladesh.

The Transcriptional Regulator TfmR Directly Regulates Two Pathogenic Pathways in Xanthomonas oryzae pv. oryzicola.

Xanthomonas oryzae pv. oryzicola (Xoc) is a notorious plant pathogen. Like most bacterial pathogens, Xoc has evolved a complex regulatory network to modulate the expression of various genes related to pathogenicity. Here, we have identified TfmR, a transcriptional regulator belonging to the TetR family, as a key player in the virulence mechanisms of this phytopathogenic bacterium. We have demonstrated genetically that tfmR is involved in the hypersensitive response (HR), pathogenicity, motility and extracellular polysaccharide production of this phytopathogenic bacterium. Our investigations extended to exploring TfmR's interaction with RpfG and HrpX, two prominent virulence regulators in Xanthomonas species. We found that TfmR directly binds to the promoter region of RpfG, thereby positively regulating its expression. Notably, constitutive expression of RpfG partly reinstates the pathogenicity compromised by TfmR-deletion mutants. Furthermore, our studies revealed that TfmR also exerts direct positive regulation on the expression of the T3SS regulator HrpX. Similar to RpfG, sustained expression of HrpX partially restores the pathogenicity of TfmR-deletion mutants. These findings underscore TfmR's multifaceted role as a central regulator governing key virulence pathways in Xoc. Importantly, our research sheds light on the intricate molecular mechanisms underlying the regulation of pathogenicity in this plant pathogen.

Transcriptome analysis reveals the inhibitory mechanism of 3,4-Dimethoxyphenol from Streptomyces albidoflavus strain ML27 against Xanthomonas oryzae pv. oryzae.

Bacterial leaf blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), poses a significant threat to rice cultivation across diverse regions. Growing concerns about pesticide resistance and environmental impact underscore the urgent necessity for eco-friendly biopesticides. Here, the complete genome sequence of Streptomyces albidoflavus strain ML27 revealed substantial antimicrobial activity and secondary metabolite production potential through genome mining. 3,4-dimethoxyphenol (purity 97%) was successfully isolated from the fermentation broth of S. albidoflavus strain ML27, exhibiting broad and pronounced inhibitory effects on the growth of seven different fungi and five tested bacteria. The efficacy of 3,4-dimethoxyphenol in controlling rice bacterial leaf blight was evaluated through pot tests, demonstrating substantial therapeutic (69.39%) and protective (84.53%) effects. Application of 3,4-dimethoxyphenol to Xoo resulted in cells displayed notable surface depressions, wrinkles, distortions, or even ruptures compared to their typical morphology. Transcriptome analysis revealed significant inhibition of membrane structures, protein synthesis and secretion, bacterial secretion system, two-component system, flagellar assembly, as well as various metabolic and biosynthetic pathways by 3,4-dimethoxyphenol. Notably, the down-regulation of the type III secretion system (T3SS) expression was a pivotal finding. Furthermore, validation via quantitative real-time polymerase chain reaction (qRT-PCR) analysis confirmed significant downregulation of 10 genes related to T3SS upon 3,4-dimethoxyphenol treatment. Based on these results, it is promising to develop 3,4-dimethoxyphenol as a novel biopesticide targeting the T3SS of Xoo for controlling bacterial leaf blight in rice.

XA21-mediated resistance to Xanthomonas oryzae pv. oryzae is dose dependent.

The rice receptor kinase XA21 confers broad-spectrum resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial blight disease. To investigate the relationship between the expression level of XA21 and resulting resistance, we generated independent HA-XA21 transgenic rice lines accumulating the XA21 immune receptor fused with an HA epitope tag. Whole-genome sequence analysis identified the T-DNA insertion sites in sixteen independent T0 events. Through quantification of the HA-XA21 protein and assessment of the resistance to Xoo strain PXO99 in six independent transgenic lines, we observed that XA21-mediated resistance is dose dependent. In contrast, based on the four agronomic traits quantified in these experiments, yield is unlikely to be affected by the expression level of HA-XA21. These findings extend our knowledge of XA21-mediated defense and contribute to the growing number of well-defined genomic landing pads in the rice genome that can be targeted for gene insertion without compromising yield.

GDP-mannose pyrophosphorylase is an efficient target in Xanthomonas citri for citrus canker control.

Xanthomonas citri subsp. citri (Xcc) is a bacterium that causes citrus canker, an economically important disease that results in premature fruit drop and reduced yield of fresh fruit. In this study, we demonstrated the involvement of XanB, an enzyme with phosphomannose isomerase (PMI) and guanosine diphosphate-mannose pyrophosphorylase (GMP) activities, in Xcc pathogenicity. Additionally, we found that XanB inhibitors protect the host against Xcc infection. Besides being deficient in motility, biofilm production, and ultraviolet resistance, the xanB deletion mutant was unable to cause disease, whereas xanB complementation restored wild-type phenotypes. XanB homology modeling allowed in silico virtual screening of inhibitors from databases, three of them being suitable in terms of absorption, distribution, metabolism, excretion, and toxicity (ADME/Tox) properties, which inhibited GMP (but not PMI) activity of the Xcc recombinant XanB protein in more than 50%. Inhibitors reduced citrus canker severity up to 95%, similarly to copper-based treatment. xanB is essential for Xcc pathogenicity, and XanB inhibitors can be used for the citrus canker control. IMPORTANCE: Xcc causes citrus canker, a threat to citrus production, which has been managed with copper, being required a more sustainable alternative for the disease control. XanB was previously found on the surface of Xcc, interacting with the host and displaying PMI and GMP activities. We demonstrated by xanB deletion and complementation that GMP activity plays a critical role in Xcc pathogenicity, particularly in biofilm formation. XanB homology modeling was performed, and in silico virtual screening led to carbohydrate-derived compounds able to inhibit XanB activity and reduce disease symptoms by 95%. XanB emerges as a promising target for drug design for control of citrus canker and other economically important diseases caused by Xanthomonas sp.

Antisense Oligonucleotide as a New Technology Application for CsLOB1 Gene Silencing Aiming at Citrus Canker Resistance.

Citrus canker disease, caused by Xanthomonas citri subsp. citri, poses a significant threat to global citrus production. The control of the disease in the field relies mainly on the use of conventional tools such as copper compounds, which are harmful to the environment and could lead to bacterial resistance. This scenario stresses the need for new and sustainable technologies to control phytopathogens, representing a key challenge in developing studies that translate basic into applied knowledge. During infection, X. citri subsp. citri secretes a transcriptional activator-like effector that enters the nucleus of plant cells, activating the expression of the canker susceptibility gene LATERAL ORGAN BOUNDARIES 1 (LOB1). In this study, we explored the use of antisense oligonucleotides (ASOs) with phosphorothioate modifications to transiently inhibit the gene expression of CsLOB1 in Citrus sinensis. We designed and validated three potential ASO sequences, which led to a significant reduction in disease symptoms compared with the control. The selected ASO3-CsLOB1 significantly decreased the expression level of CsLOB1 when delivered through two distinct delivery methods, and the reduction of the symptoms ranged from approximately 15 to 83%. Notably, plants treated with ASO3 did not exhibit an increase in symptom development over the evaluation period. This study highlights the efficacy of ASO technology, based on short oligonucleotide chemically modified sequences, as a promising tool for controlling phytopathogens without the need for genetic transformation or plant regeneration. Our results demonstrate the potential of ASOs as a biotechnological tool for the management of citrus canker disease.

Genome analysis and classification of Xanthomonas bacteriophage AhaSv, a new member of the genus Salvovirus.

Xanthomonas phage AhaSv was isolated from lake water. Genome sequencing showed that its genome is a linear dsDNA molecule with a length of 55,576 bp and a G+C content of 63.23%. Seventy-one open reading frames (ORFs) were predicted, and no tRNAs were found in the genome. Phylogenetic analysis showed that AhaSv is closely related to members of the genus Salvovirus of the family Casjensviridae. Intergenomic similarity values between phage AhaSv and homologous phages were up to 90.6%, suggesting that phage AhaSv should be considered a member of a new species in the genus Salvovirus.