Study of cabbage antioxidant system response on early infection stage of Xanthomonas campestris pv. campestris.

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc) significantly affects the production of cabbage and other cruciferous vegetables. Plant antioxidant system plays an important role in pathogen invasion and is one of the main mechanisms underlying resistance to biological stress. Therefore, it is important to study the resistance mechanisms of the cabbage antioxidant system during the early stages of Xcc. In this study, 108 CFU/mL (OD600 = 0.1) Xcc race1 was inoculated on "zhonggan 11" cabbage using the spraying method. The effects of Xcc infection on the antioxidant system before and after Xcc inoculation (0, 1, 3, and 5 d) were studied by physiological indexes determination, transcriptome and metabolome analyses. We concluded that early Xcc infection can destroy the balance of the active oxygen metabolism system, increase the generation of free radicals, and decrease the scavenging ability, leading to membrane lipid peroxidation, resulting in the destruction of the biofilm system and metabolic disorders. In response to Xcc infection, cabbage clears a series of reactive oxygen species (ROS) produced during Xcc infection via various antioxidant pathways. The activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) increased after Xcc infection, and the ROS scavenging rate increased. The biosynthesis of non-obligate antioxidants, such as ascorbic acid (AsA) and glutathione (GSH), is also enhanced after Xcc infection. Moreover, the alkaloid and vitamin contents increased significantly after Xcc infection. We concluded that cabbage could resist Xcc invasion by maintaining the stability of the cell membrane system and improving the biosynthesis of antioxidant substances and enzymes after infection by Xcc. Our results provide theoretical basis and data support for subsequent research on the cruciferous vegetables resistance mechanism and breeding to Xcc.

Incorporation of engineered nanoparticles of biochar and fly ash against bacterial leaf spot of pepper.

In agriculture, the search for higher net profit is the main challenge in the economy of the producers and nano biochar attracts increasing interest in recent years due to its unique environmental behavior and increasing the productivity of plants by inducing resistance against phytopathogens. The effect of rice straw biochar and fly ash nanoparticles (RSBNPs and FNPs, respectively) in combination with compost soil on bacterial leaf spot of pepper caused by Xanthomonas campestris pv. vesicatoria was investigated both in vitro and in vivo. The application of nanoparticles as soil amendment significantly improved the chili pepper plant growth. However, RSBNPs were more effective in enhancing the above and belowground plant biomass production. Moreover, both RSBNPs and FNPs, significantly reduced (30.5 and 22.5%, respectively), while RSBNPs had shown in vitro growth inhibition of X. campestris pv. vesicatoria by more than 50%. The X-ray diffractometry of RSBNPs and FNPs highlighted the unique composition of nano forms which possibly contributed in enhancing the plant defence against invading X. campestris pv. vesicatoria. Based on our findings, it is suggested that biochar and fly ash nanoparticles can be used for reclaiming the problem soil and enhance crop productivity depending upon the nature of the soil and the pathosystem under investigation.

The lolB gene in Xanthomonas campestris pv. campestris is required for bacterial attachment, stress tolerance, and virulence.

BACKGROUND: Xanthomonas campestris pv. campestris (Xcc) is a Gram-negative bacterium that can cause black rot disease in crucifers. The lipoprotein outer membrane localization (Lol) system is involved in the lipoprotein sorting to the outer membrane. Although Xcc has a set of annotated lol genes, there is still little known about the physiological role in this phytopathogen. In this study, we aimed to characterize the role of LolB of Xcc in bacterial attachment, stress tolerance, and virulence. RESULTS: To characterize the role of LolB, lolB mutant was constructed and phenotypic evaluation was performed. The lolB mutant revealed reductions in bacterial attachment, extracellular enzyme production, and virulence. Mutation of lolB also resulted in reduced tolerance to a myriad of stresses, including heat and a range of membrane-perturbing agents. Trans-complementation of lolB mutant with intact lolB gene reverted these altered phenotypes to the wild-type levels. From subsequent reporter assay and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis, the expression of genes that encode the major extracellular enzymes and the stress-related proteins was reduced after lolB mutation. CONCLUSIONS: The results in this work contribute to the functional understanding of lolB in Xanthomonas for the first time, and provide new insights into the function of lolB in bacteria.

Potentiation of plant defense by bacterial outer membrane vesicles is mediated by membrane nanodomains.

Outer membrane vesicles (OMVs) are released from the outer membranes of Gram-negative bacteria during infection and modulate host immunity during host-pathogen interactions. The mechanisms by which OMVs are perceived by plants and affect host immunity are unclear. Here, we used the pathogen Xanthomonas campestris pv. campestris to demonstrate that OMV-plant interactions at the Arabidopsis thaliana plasma membrane (PM) modulate various host processes, including endocytosis, innate immune responses, and suppression of pathogenesis by phytobacteria. The lipid phase of OMVs is highly ordered and OMVs directly insert into the Arabidopsis PM, thereby enhancing the plant PM's lipid order; this also resulted in strengthened plant defenses. Strikingly, the integration of OMVs into the plant PM is host nanodomain- and remorin-dependent. Using coarse-grained simulations of molecular dynamics, we demonstrated that OMV integration into the plant PM depends on the membrane lipid order. Our computational simulations further showed that the saturation level of the OMV lipids could fine-tune the enhancement of host lipid order. Our work unraveled the mechanisms underlying the ability of OMVs produced by a plant pathogen to insert into the host PM, alter host membrane properties, and modulate plant immune responses.

Predatory and biocontrol potency of Bdellovibrio bacteriovorus toward phytopathogenic strains of Pantoea sp. and Xanthomonas campestris in the presence of exo-biopolymers: in vitro and in vivo assessments.

Bdellovibrios are predatory bacteria that invade other live Gram-negative bacterial cells for growth and reproduction. They have recently been considered as potential living antibiotics and biocontrol agents. In this study, the predatory activity and biocontrol potency of Bdellovibrio bacteriovorus strain SOIR-1 against Pantoea sp. strain BCCS and Xanthomonas campestris, two exo-biopolymer-producing phytopathogens, was evaluated. Plaque formation assays and lysis analysis in the broth co-cultures were used for the in vitro evaluation of bacteriolytic activity of strain SOIR-1. The in vivo biocontrol potential of strain SOIR-1 was evaluated by pathogenicity tests on the onion bulbs and potato tuber slices. The phytopathogens were also recovered from the infected plant tissues and confirmed using biochemical tests and PCR-based 16S rRNA gene sequence analysis. Typical bdellovibrios plaques were developed on the lawn cultures of Pantoea sp. BCCS and X. campestris. The killing rate of strain SOIR-1 toward Pantoea sp. BCCS and X. campestris was 84.3% and 76.3%, respectively. Exo-biopolymers attenuated the predation efficiency of strain SOIR-1 up to 10.2-18.2% (Pantoea sp. BCCS) and 12.2-17.3% (X. campestris). The strain SOIR-1 significantly reduced rotting symptoms in the onion bulbs caused by Pantoea sp. BCCS (69.0%) and potato tuber slices caused by X. campestris (73.1%). Although more field assessments are necessary, strain SOIR-1 has the preliminary potential as a biocontrol agent against phytopathogenic Pantoea sp. BCCS and X. campestris, especially in postharvest storage. Due to the particular physicochemical properties of evaluated exo-biopolymers, they can be used in the designing encapsulation systems for delivery of bdellovibrios.

Pr-favoured variants of the bacteriophytochrome from the plant pathogen Xanthomonas campestris hint on light regulation of virulence-associated mechanisms.

Red/far-red light-sensing bacteriophytochrome photoreceptor (BphP) pathways play key roles in bacterial physiology and ecology. These bilin-binding proteins photoswitch between two states, Pr (red absorbing) and Pfr (far-red absorbing). The isomerization of the chromophore and the downstream structural changes result in the light signal transduction. The agricultural pathogen Xanthomonas campestris pv. campestris (Xcc) code for a single bathy-like type BphP (XccBphP), previously shown to negatively regulate several light-mediated biological processes involved in virulence. Here, we generated three different full-length variants with single amino acid changes within its GAF domain that affect the XccBphP photocycle favouring its Pr state: L193Q, L193N and D199A. While D199A recombinant protein locks XccBphP in a Pr-like state, L193Q and L193N exhibit a significant enrichment of the Pr form in thermal equilibrium. The X-ray crystal structures of the three variants were solved, resembling the wild-type protein in the Pr state. Finally, we studied the effects of altering the XccBphP photocycle on the exopolysaccharide xanthan production and stomatal aperture assays as readouts of its bacterial signalling pathway. Null-mutant complementation assays show that the photoactive Pr-favoured XccBphP variants L193Q and L193N tend to negatively regulate xanthan production in vivo. In addition, our results indicate that strains expressing these variants also promote stomatal apertures in challenged plant epidermal peels, compared to wild-type Xcc. The findings presented in this work provide new evidence on the Pr state of XccBphP as a negative regulator of the virulence-associated mechanisms by light in Xcc.

An ERF121 transcription factor from Brassica oleracea is a target for the conserved TAL-effectors from different Xanthomonas campestris pv. campestris strains.

Transcription activator-like effectors (TALEs), which induce the expression of specific plant genes to promote infection, are the main pathogenic determinants of various Xanthomonas bacteria. However, investigation of TALEs from Xanthomonas campestris pv. campestris, which causes black rot disease of crucifers, received little attention. In this study, we used PCR-based amplification followed by SMRT amplicon sequencing to identify TALE genes in several X. campestris pv. campestris strains. Computational prediction in conjunction with quantitative reverse transcription PCR analysis was used to find their targets in the Brassica oleracea genome. Transcription factor ERF121, from the AP2/ERF family, was identified as target gene for the conserved TALEs from multiple X. campestris pv. campestris strains. Several members of this family from diverse plants were previously identified as targets of TALEs from different Xanthomonas species. We propose that TALE-dependent activation of AP2/ERF transcription factors promotes susceptibility to Xanthomonas through the misregulation of plant defence pathways.

PilG and PilH antagonistically control flagellum-dependent and pili-dependent motility in the phytopathogen Xanthomonas campestris pv. campestris.

BACKGROUND: The virulence of the plant pathogen Xanthomonas campestris pv. campestris (Xcc) involves the coordinate expression of many virulence factors, including surface appendages flagellum and type IV pili, which are required for pathogenesis and the colonization of host tissues. Despite many insights gained on the structure and functions played by flagellum and pili in motility, biofilm formation, surface attachment and interactions with bacteriophages, we know little about how these appendages are regulated in Xcc. RESULTS: Here we present evidence demonstrating the role of two single domain response regulators PilG and PilH in the antagonistic control of flagellum-dependent (swimming) and pili-dependent (swarming) motility. Using informative mutagenesis, we reveal PilG positively regulates swimming motility while and negatively regulating swarming motility. Conversely, PilH negatively regulates swimming behaviour while and positively regulating swarming motility. By transcriptome analyses (RNA-seq and RT-PCR) we confirm these observations as PilG is shown to upregulate many genes involved chemotaxis and flagellar biosynthesis but these similar genes were downregulated by PilH. Co-immunoprecipitation, bacterial two-hybrid and pull-down analyses showed that PilH and PilG were able to interact with district subsets of proteins that potentially account for their regulatory impact. Additionally, we present evidence, using mutagenesis that PilG and PilH are involved in other cellular processes, including chemotaxis and virulence. CONCLUSIONS: Taken together, we demonstrate that for the conditions tested PilG and PilH have inverse regulatory effects on flagellum-dependent and pili-dependent motility in Xcc and that this regulatory impact depends on these proteins influences on genes/proteins involved in flagellar biosynthesis and pilus assembly.

Chemical Targeting and Manipulation of Type III Secretion in the Phytopathogen Xanthomonas campestris for Control of Disease.

Xanthomonas campestris pv. campestris is the causative agent of black rot disease in crucifer plants. This Gram-negative bacterium utilizes the type III secretion system (T3SS), encoded by the hrp gene cluster, to aid in its resistance to host defenses and the ability to cause disease. The T3SS injects a set of proteins known as effectors into host cells that come into contact with the bacterium. The T3SS is essential for the virulence and hypersensitive response (HR) of X. campestris pv. campestris, making it a potential target for disease control strategies. Using a unique and straightforward high-throughput screening method, we examined a large collection of diverse small molecules for their potential to modulate the T3SS without affecting the growth of X. campestris pv. campestris. Screening of 13,129 different compounds identified 10 small molecules that had a significant inhibitory influence on T3SS. Moreover, reverse transcription-quantitative PCR (qRT-PCR) assays demonstrated that all 10 compounds repress the expression of the hrp genes. Interestingly, the effect of these small molecules on hrp genes may be through the HpaS and ColS sensor kinase proteins that are key to the regulation of the T3SS in planta Five of the compounds were also capable of inhibiting X. campestris pv. campestris virulence in a Chinese radish leaf-clipping assay. Furthermore, seven of the small molecules significantly weakened the HR in nonhost pepper plants challenged with X. campestris pv. campestris. Taken together, these small molecules may provide potential tool compounds for the further development of antivirulence agents that could be used in disease control of the plant pathogen X. campestris pv. campestris.IMPORTANCE The bacterium Xanthomonas campestris pv. campestris is known to cause black rot disease in many socioeconomically important vegetable crops worldwide. The management and control of black rot disease have been tackled with chemical and host resistance methods with variable success. This has motivated the development of alternative methods for preventing this disease. Here, we identify a set of novel small molecules capable of inhibiting X. campestris pv. campestris virulence, which may represent leading compounds for the further development of antivirulence agents that could be used in the control of black rot disease.

The clpX gene plays an important role in bacterial attachment, stress tolerance, and virulence in Xanthomonas campestris pv. campestris.

Xanthomonas campestris pv. campestris is a bacterial pathogen and the causal agent of black rot in crucifers. In this study, a clpX mutant was obtained by EZ-Tn5 transposon mutagenesis of the X. campestris pv. campestris. The clpX gene was annotated to encode ClpX, the ATP-binding subunit of ATP-dependent Clp protease. The clpX mutant exhibited reduced bacterial attachment, extracellular enzyme production and virulence. Mutation of clpX also resulted in increased sensitivity to a myriad of stresses, including heat, puromycin, and sodium dodecyl sulfate. These altered phenotypes of the clpX mutant could be restored to wild-type levels by in trans expression of the intact clpX gene. Proteomic analysis revealed that the expression of 211 proteins differed not less than twofold between the wild-type and mutant strains. Cluster of orthologous group analysis revealed that these proteins are mainly involved in metabolism, cell wall biogenesis, chaperone, and signal transduction. The reverse transcription quantitative real-time polymerase chain reaction analysis demonstrated that the expression of genes encoding attachment-related proteins, extracellular enzymes, and virulence-associated proteins was reduced after clpX mutation. The results in this study contribute to the functional understanding of the role of clpX in Xanthomonas for the first time, and extend new insights into the function of clpX in bacteria.

The roles of histidine kinases in sensing host plant and cell-cell communication signal in a phytopathogenic bacterium.

It has long been known that phytopathogenic bacteria react to plant-specific stimuli or environmental factors. However, how bacterial cells sense these environmental cues remains incompletely studied. Recently, three kinds of histidine kinases (HKs) were identified as receptors to perceive plant-associated or quorum-sensing signals. Among these kinases, HK VgrS detects iron depletion by binding to ferric iron via an ExxE motif, RpfC binds diffusible signal factor (DSF) by its N-terminal peptide and activates its autokinase activity through relaxation of autoinhibition, and PcrK specifically senses plant hormone-cytokinin and elicits bacterial responses to oxidative stress. These HKs are critical sensors that regulate the virulence of a Gram-negative bacterium, Xanthomonas campestris pv. campestris. Research progress on the signal perception of phytopathogenic bacterial HKs suggests that inter-kingdom signalling between host plants and pathogens controls pathogenesis and can be used as a potential molecular target to protect plants from bacterial diseases. This article is part of the theme issue 'Biotic signalling sheds light on smart pest management'.

Two lytic transglycosylases of Xanthomonas campestris pv. campestris associated with cell separation and type III secretion system, respectively.

The lytic transglycosylases (LTs) are important enzymes that degrade peptidoglycan of the bacterial cell wall and affect many biological functions. We present here that XC_0706 and XC_3001 are annotated as the LTs in Xanthomonas campestris pv. campestris. XC_0706 is associated with virulence and plays a pivotal role in cell division. Mutation on XC_3001 reduced hypersensitive response induction and the translocation of type III effector, but did not affect the function of the type II secretion system. Further studies showed that multiple LTs genes contribute to efficiency of the type III secretory system in X. campestris pv. campestris.

Dissecting quantitative resistance to Xanthomonas campestris pv. campestris in leaves of Brassica oleracea by QTL analysis.

Black rot, caused by the bacterium Xanthomonas campestris pv. campestris (Xcc), produces important economic losses in crops of Brassica oleracea worldwide. Resistance to race 1, the most virulent and widespread in B. oleracea, is under quantitative control. Knowledge about the genetics of this resistance would help in designing strategies to control initial stages of invasion and development of the disease. QTL analysis of the resistance in the BolTBDH mapping population was performed. Resistance was measured with five traits related to initial stages of the invasion, success of infection and spread of the pathogen. Four single-trait QTLs of resistance were found, from which one represent novel variation. After performing multi-trait QTL, we concluded that spread of Xcc is related to the size of the leaf. Individuals from the mapping population follow two different strategies to cope with the spread of the disease: reducing lesion size or maintain more area of the leaf photosynthetically active, being more tolerant to Xcc invasion. Mechanisms underlying variation for resistance may be related to different aspects of plant immunity, including the synthesis of glucosinolates and phenolics.

BDSF Is the Predominant In-Planta Quorum-Sensing Signal Used During Xanthomonas campestris Infection and Pathogenesis in Chinese Cabbage.

Xanthomonas campestris pv. campestris uses the diffusible signal factor (DSF) family of quorum-sensing (QS) signals to coordinate virulence and adaptation. DSF family signals have been well-characterized using laboratory-based cell cultures. The in-planta QS signal used during X. campestris pv. campestris infection remains unclear. To achieve this goal, we first mimic in-planta X. campestris pv. campestris growth conditions by supplementing the previously developed XYS medium with cabbage hydrolysate and found that the dominant signal produced in these conditions was BDSF. Secondly, by using XYS medium supplemented with diverse plant-derived compounds, we examined the effects of diverse plant-derived compounds on the biosynthesis of DSF family signals. Several compounds were found to promote biosynthesis of BDSF. Finally, using an X. campestris pv. campestris ΔrpfB-Chinese cabbage infection model and an ultra-performance liquid chromatographic-time of flight-mass spectrometry-based assay, BDSF was found to comprise >70% of the DSF family signals present in infected cabbage tissue. BDSF at a concentration of 2.0 µM induced both protease activity and engXCA expression. This is the first report to directly show that BDSF is the predominant in-planta QS signal used during X. campestris pv. campestris infection. It provides a better understanding of the molecular interactions between X. campestris pv. campestris and its cruciferous hosts and also provides the logical target for designing strategies to counteract BDSF signaling and, thus, infection. Further studies are needed to get an exact idea about the DSF production dynamics of the wild-type strain inside the plant.

The RNA chaperone Hfq is important for the virulence, motility and stress tolerance in the phytopathogen Xanthomonas campestris.

The RNA chaperone, Hfq, is known to play extensive roles in bacterial growth and development. More recently, it has been shown to be required for virulence in many human and animal bacterial pathogens. Despite these studies little is known about the role Hfq plays in phytopathogenic bacteria. In this study, we show Hfq is required for full virulence of the crucifer black rot pathogen Xanthomonas campestris pv. campestris (Xcc). We demonstrate that an Xcc hfq deletion strain is highly attenuated for virulence in Chinese radish and shows a severe defect in the production of virulence factors including extracellular enzymes and extracellular polysaccharide. Furthermore, the Xcc strain lacking Hfq had significantly reduced cell motility and stress tolerance. These findings suggest that Hfq is a key regulator of important aspects of virulence and adaptation of Xcc. Taken together, our findings are suggestive of a regulatory network placing Hfq at the centre of virulence gene expression control in Xcc.

Heterologous expression and antimicrobial activity of OsGASR3 from rice (Oryza sativa L.).

According to an in silico analysis, OsGASR3 (LOC_Os03g55290) from rice (Oryza sativa L.) was predicted to be involved in plant defense mechanisms. A semi-quantitative reverse transcription polymerase chain reaction assay revealed that OsGASR3 is highly expressed in the inflorescences of Thai jasmine rice (O. sativa L. subsp. indica 'KDML 105'). To characterize the biological activity of OsGASR3, we produced an OsGASR3-glutathione S-transferase fusion protein in Escherichia coli Rosetta-gami (DE3) cells for a final purified recombinant OsGASR3 yield of 0.65 mg/L. The purified OsGASR3 inhibited the hyphal growth of Fusarium oxysporum f.sp. cubense and Helminthosporium oryzae at a relatively low concentration (7.5 µg/mL). Furthermore, OsGASR3 exhibited in planta inhibitory activity against Xanthomonas campestris, suggesting its involvement in defense mechanisms, in addition to its previously reported functions affecting growth and development. These observations indicate that recombinant OsGASR3 may be useful for protecting agriculturally important crops against pathogenic microbes.

Unraveling the metabolic response of Brassica oleracea exposed to Xanthomonas campestris pv. campestris.

BACKGROUND: Brassica crops together with cereals represent the basis of world supplies. Due to their importance, the production losses caused by Xanthomonas campestris pv. campestris (Xcc) infection represent a high economic impact. Understanding molecular and biochemical mechanisms of plants is essential to develop resistant crops with durable protection against diseases. In this regard, metabolomics has emerged as a valuable technology to provide an overview of the biological status of a plant exposed to a disease. This study investigated the dynamic changes in the metabolic profile of Brassica oleracea plants during an Xcc infection from leaves collected at five different days post infection using a mass spectrometry approach. RESULTS: Results showed that Xcc infection causes dynamic changes in the metabolome of B. oleracea. Moreover, induction/repression pattern of the metabolites implicated in the response follows a complex dynamics during infection progression, indicating a complex temporal response. Specific metabolic pathways such as alkaloids, coumarins or sphingolipids are postulated as promising key role candidates in the infection response. CONCLUSION: This work tries to decipher the changes produced on Brassica crops metabolome under Xcc infection and represents a step forward in the understanding of B. oleracea-Xcc interaction. © 2018 Society of Chemical Industry.

A 1-phytase type III effector interferes with plant hormone signaling.

Most Gram-negative phytopathogenic bacteria inject type III effector (T3E) proteins into plant cells to manipulate signaling pathways to the pathogen's benefit. In resistant plants, specialized immune receptors recognize single T3Es or their biochemical activities, thus halting pathogen ingress. However, molecular function and mode of recognition for most T3Es remains elusive. Here, we show that the Xanthomonas T3E XopH possesses phytase activity, i.e., dephosphorylates phytate (myo-inositol-hexakisphosphate, InsP6), the major phosphate storage compound in plants, which is also involved in pathogen defense. A combination of biochemical approaches, including a new NMR-based method to discriminate inositol polyphosphate enantiomers, identifies XopH as a naturally occurring 1-phytase that dephosphorylates InsP6 at C1. Infection of Nicotiana benthamiana and pepper by Xanthomonas results in a XopH-dependent conversion of InsP6 to InsP5. 1-phytase activity is required for XopH-mediated immunity of plants carrying the Bs7 resistance gene, and for induction of jasmonate- and ethylene-responsive genes in N. benthamiana.

Functional characterization and transcriptional analysis of icd2 gene encoding an isocitrate dehydrogenase of Xanthomonas campestris pv. campestris.

Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate to alpha-ketoglutarate. In the genome of Xanthomonas campestris pv. campestris, the phytopathogen that causes black rot in cruciferous plants, two putative IDH genes, icd1 and icd2, have been annotated. Their physiological roles in X. campestris pv. campestris are unclear. In this study, the icd2 gene from X. campestris pv. campestris was characterized in detail. We demonstrated genetically that icd2 gene encodes a functional IDH, and is involved in virulence as well as bacterial attachment. Furthermore, the icd2 transcription initiation site was mapped at nucleotide G, 127 nucleotide upstream of the icd2 translation start codon. In addition, promoter analysis revealed that icd2 expression exhibits a distinct expression profile under different culture conditions, is subjected to catabolite repression, and is affected by acetate. This is the first time that the function and transcription of icd2 have been characterized in the crucifer pathogen X. campestris pv. campestris.