Gram-negative Phytopathogenic Bacteria Research Articles

Phytochemical Study of Ethanol Extract of Gnaphalium uliginosum L. and Evaluation of Its Antimicrobial Activity.

This study evaluates the antibacterial and antifungal effects of ethanol extracts from Gnaphalium uliginosum L. derived from freshly harvested plant biomass, including stems, leaves, flowers, and roots. The extract was analyzed using gas chromatography-mass spectrometry (GC-MS) to determine its antimicrobial activity against phytopathogenic bacteria and fungi. Two methods were used in the experiments: agar well diffusion and double serial dilution. Extraction was carried out using the maceration method with different temperature regimes (25 °C, 45 °C, and 75 °C) and the ultrasonic method at various powers (63-352 W) for different durations (5 and 10 min). It was found that the 70% ethanol extract obtained through the ultrasonic experiment at 189 W power for 10 min and at 252 W power for 5 min had the highest antimicrobial activity compared to the maceration method. The most sensitive components of the extracts were the Gram-positive phytopathogenic bacteria Clavibacter michiganensis and the Gram-negative phytopathogenic bacteria Erwinia carotovora spp., with MIC values of 156 μg/mL. Among the fungi, the most sensitive were Rhizoctonia solani and Alternaria solani (MIC values in the range of 78-156 µg/mL). The evaluation of the antimicrobial activity of extracts using the diffusion method established the presence of a growth suppression zone in the case of C. michiganensis (15-17 mm for flowers, leaves, and total biomass), which corresponds to the average level of antimicrobial activity. These findings suggest that G. uliginosum has potential as a source of biologically active compounds for agricultural use, particularly for developing novel biopesticides.

Structural insights into Xanthomonas campestris pv. campestris NAD+ biosynthesis via the NAM salvage pathway

Nicotinamide phosphoribosyltransferase (NAMPT) plays an important role in the biosynthesis of nicotinamide adenine dinucleotide (NAD+) via the nicotinamide (NAM) salvage pathway. While the structural biochemistry of eukaryote NAMPT has been well studied, the catalysis mechanism of prokaryote NAMPT at the molecular level remains largely unclear. Here, we demonstrated the NAMPT-mediated salvage pathway is functional in the Gram-negative phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc) for the synthesis of NAD+, and the enzyme activity of NAMPT in this bacterium is significantly higher than that of human NAMPT in vitro. Our structural analyses of Xcc NAMPT, both in isolation and in complex with either the substrate NAM or the product nicotinamide mononucleotide (NMN), uncovered significant details of substrate recognition. Specifically, we revealed the presence of a NAM binding tunnel that connects the active site, and this tunnel is essential for both catalysis and inhibitor binding. We further demonstrated that NAM binding in the tunnel has a positive cooperative effect with NAM binding in the catalytic site. Additionally, we discovered that phosphorylation of the His residue at position 229 enhances the substrate binding affinity of Xcc NAMPT and is important for its catalytic activity. This work reveals the importance of NAMPT in bacterial NAD+ synthesis and provides insights into the substrate recognition and the catalytic mechanism of bacterial type II phosphoribosyltransferases.

Sustainable green synthesis of Hedychium coronarium leaf extract-stabilized silver nanoparticles and their applications: colorimetric sensing of Sn2+ and Hg2+ and antifungal and antimicrobial properties.

Hedychium coronarium (Hc) (commonly known as Gulbakawali) leaf extract was used for the stable and sustainable green synthesis of silver nanoparticles (Hc-AgNPs), which were biodegradable and non-toxic. Hedychium coronarium leaf extract was used as a reducing agent to stabilize the Hc-AgNPs by converting Ag+ to Ag0 without adding any capping agent. It demonstrated stability for up to six months, and no agglomeration was observed. The Hc-AgNPs were characterized using X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), ultraviolet-visible spectrophotometry, and fluorescence spectroscopy analysis. The UV-visible spectrum supported the formation of stable Hc-AgNPs by displaying a strong surface plasmon resonance (SPR) peak at 440 nm. FT-IR spectra showed the functional groups present in the leaf extract of Hedychium coronarium, which was the primary source of secondary metabolites attached to Ag0. XRD analysis revealed a distinct 2θ peak of Hc-AgNPs at 38.15°, indicating a face-centred cubic structure with a crystallite size of 22.6 ± 1 nm at the (111) plane. Moreover, TEM demonstrated the spherical morphology of the Hc-AgNPs with an average particle size of 22.42 ± 1 nm. The photophysical characteristics of the Hc-AgNPs, as highlighted by their UV-vis and fluorescence characteristics, revealed their semiconducting nature with an impressive band gap (E g) value of 3.78 eV. Fascinatingly, the fluorescence activity of Hc-AgNPs at 504 nm showed a broad emission band corresponding to the absorption band at 251 nm. We performed the selective colorimetric sensing of Sn2+ metal ions using Hc-AgNPs, which demonstrated a detection limit of 10-3 M, suggesting their potential as very good solid biosensors. Interestingly, the Hc-AgNPs showed antifungal activity, which has not been reported before. Specifically, the results showed that the Hc-AgNPs had a higher fungitoxicity effect against Aspergillus flavus (59.58 ± 3.68) than against Fusarium oxysporum (57.93 ± 4.18). The antibacterial activity of the Hc-AgNPs was evaluated against three Gram-negative phytopathogenic bacteria: Xanthomonas oryzae (X. oryzae), Ralstonia solanacearum (R. solanacearum), and Erwinia carotovora (E. carotovora), showing effective inhibition zones of 16.33 ± 0.57, 15.33 ± 0.57, and 14.33 ± 0.57 mm, respectively. These results indicate that the Hc-AgNPs could inhibit these phytopathogenic bacteria with varying degrees of effectiveness in the order of X. oryzae > R. solanacearum > E. carotovora.

Methods of identification of phytopathogenic bacteria Pectobacterium carotovorum subsp. carotovorum

Bacterial infections are a global problem. The most common pathogens of infections among gram-negative bacteria are representatives of the Enterobacteriaceae family. Pectobacterium are gram-negative phytopathogenic bacteria belonging to the Enterobacteriaceae family.The purpose of the study is to develop methods for the identification of phytopathogenic bacteria. At the capacity of test components of developed identification algorithm, we used the data presented in the reference «Bergey’s Manual of Systematics of Archaea and Bacteria». research parameters and for selection of Model microorganisms bacteriological tests were reference strains of Pectobacterium carotovorum B-3455 obtained from the All-Russian collection of microorganisms and Pectobacterium carotovorum 333 from the collection of NRCEM Museum of FSBEI HE Ulyanovsk SAU named after P.А. Stolypin. From 50 samples of phytosanitary control and environmental objects, 5 strains were classified as Pectobacterium carotovorum spp. carotovorum.

Unleashing the Influence of cAMP Receptor Protein: The Master Switch of Bacteriocin Export in Pectobacterium carotovorum subsp. carotovorum.

Pectobacterium carotovorum subsp. carotovorum (Pcc) is a Gram-negative phytopathogenic bacterium that produces carocin, a low-molecular-weight bacteriocin that can kill related strains in response to factors in the environment such as UV exposure or nutritional deficiency. The function of the catabolite activator protein (CAP), also known as the cyclic AMP receptor protein (CRP), as a regulator of carocin synthesis was examined. The crp gene was knocked out as part of the investigation, and the outcomes were assessed both in vivo and in vitro. Analysis of the DNA sequence upstream of the translation initiation site of carocin S3 revealed two putative binding sites for CRP that were confirmed using a biotinylated probe pull-down experiment. This study revealed that the deletion of crp inhibited genes involved in extracellular bacteriocin export via the flagellar type III secretion system and impacted the production of many low-molecular-weight bacteriocins. The biotinylated probe pull-down test demonstrated that when UV induction was missing, CRP preferentially attached to one of the two CAP sites while binding to both when UV induction was present. In conclusion, our research aimed to simulate the signal transduction system that controls the expression of the carocin gene in response to UV induction.

Molecular basis for host responses to Xanthomonas infection.

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.

The Role of RelA and SpoT on ppGpp Production, Stress Response, Growth Regulation, and Pathogenicity in Xanthomonas campestris pv. campestris.

ABSTRACTThe alarmone ppGpp plays an important role in the survival of bacteria by triggering the stringent response when exposed to environmental stress. Although Xanthomonas campestris pv. campestris (Xcc), which causes black rot disease in crucifers, is a representative species of Gram-negative phytopathogenic bacteria, relatively little is known regarding the factors influencing the stringent response in this species. However, previous studies in other Gram-negative bacteria have indicated that RelA and SpoT play a critical role in ppGpp synthesis. The current study found that these proteins also had an important role in Xcc, with a ΔrelAΔspoT double mutant being unable to produce ppGpp, resulting in changes to phenotype including reduced production of exopolysaccharides (EPS), exoenzymes, and biofilm, as well the loss of swarming motility and pathogenicity. The ppGpp-deficient mutant also exhibited greater sensitivity to environment stress, being almost incapable of growth on modified minimal medium (mMM) and having a much greater propensity to enter the viable but nonculturable (VBNC) state in response to oligotrophic conditions (0.85% NaCl). These findings much advance our understanding of the role of ppGpp in the biology of Xcc and could have important implications for more effective management of this important pathogen.IMPORTANCE Xanthomonas campestris pv. campestris (Xcc) is a typical seedborne phytopathogenic bacterium that causes large economic losses worldwide, and this is the first original research article to investigate the role of ppGpp in this important species. Here, we revealed the function of RelA and SpoT in ppGpp production, physiology, pathogenicity, and stress resistance in Xcc. Most intriguingly, we found that ppGpp levels and downstream ppGpp-dependent phenotypes were mediated predominantly by SpoT, with RelA having only a supplementary role. Taken together, the results of the current study provide new insight into the role of ppGpp in the biology of Xcc, which could also have important implications for the role of ppGpp in the survival and pathogenicity of other pathogenic bacteria.

Two DNA Methyltransferases for Site-Specific 6mA and 5mC DNA Modification in Xanthomonas euvesicatoria.

Xanthomonas euvesicatoria (Xe) is a gram-negative phytopathogenic bacterium that causes bacterial spot disease in tomato/pepper leading to economic losses in plantations. DNA methyltransferases (MTases) are critical for the survival of prokaryotes; however, their functions in phytopathogenic bacteria remain unclear. In this study, we characterized the functions of two putative DNA MTases, XvDMT1 and XvDMT2, in Xe by generating XvDMT1- and XvDMT2-overexpressing strains, Xe(XvDMT1) and Xe(XvDMT2), respectively. Virulence of Xe(XvDMT2), but not Xe(XvDMT1), on tomato was dramatically reduced. To postulate the biological processes involving XvDMTs, we performed a label-free shotgun comparative proteomic analysis, and results suggest that XvDMT1 and XvDMT2 have distinct roles in Xe. We further characterized the functions of XvDMTs using diverse phenotypic assays. Notably, both Xe(XvDMT1) and Xe(XvDMT2) showed growth retardation in the presence of sucrose and fructose as the sole carbon source, with Xe(XvDMT2) being the most severely affected. In addition, biofilm formation and production of exopolysaccharides were declined in Xe(XvDMT2), but not Xe(XvDMT1). Xe(XvDMT2) was more tolerant to EtOH than Xe(XvDMT1), which had enhanced tolerance to sorbitol but decreased tolerance to polymyxin B. Using single-molecule real-time sequencing and methylation-sensitive restriction enzymes, we successfully predicted putative motifs methylated by XvDMT1 and XvDMT2, which are previously uncharacterized 6mA and 5mC DNA MTases, respectively. This study provided new insights into the biological functions of DNA MTases in prokaryotic organisms.

Bacteriocins Targeting Gram-Negative Phytopathogenic Bacteria: Plantibiotics of the Future.

Gram-negative phytopathogenic bacteria are a significant threat to food crops. These microbial invaders are responsible for a plethora of plant diseases and can be responsible for devastating losses in crops such as tomatoes, peppers, potatoes, olives, and rice. Current disease management strategies to mitigate yield losses involve the application of chemicals which are often harmful to both human health and the environment. Bacteriocins are small proteinaceous antibiotics produced by bacteria to kill closely related bacteria and thereby establish dominance within a niche. They potentially represent a safer alternative to chemicals when used in the field. Bacteriocins typically show a high degree of selectivity toward their targets with no off-target effects. This review outlines the current state of research on bacteriocins active against Gram-negative phytopathogenic bacteria. Furthermore, we will examine the feasibility of weaponizing bacteriocins for use as a treatment for bacterial plant diseases.

Plant-Derived Inhibitors of AHL-Mediated Quorum Sensing in Bacteria: Modes of Action.

Numerous gram-negative phytopathogenic and zoopathogenic bacteria utilise acylated homoserine lactone (AHL) in communication systems, referred to as quorum sensing (QS), for induction of virulence factors and biofilm development. This phenomenon positions AHL-mediated QS as an attractive target for anti-infective therapy. This review focused on the most significant groups of plant-derived QS inhibitors and well-studied individual compounds for which in silico, in vitro and in vivo studies provide substantial knowledge about their modes of anti-QS activity. The current data about sulfur-containing compounds, monoterpenes and monoterpenoids, phenylpropanoids, benzoic acid derivatives, diarylheptanoids, coumarins, flavonoids and tannins were summarized; their plant sources, anti-QS effects and bioactivity mechanisms have also been summarized and discussed. Three variants of plant-derived molecules anti-QS strategies are proposed: (i) specific, via binding with LuxI-type AHL synthases and/or LuxR-type AHL receptor proteins, which have been shown for terpenes (carvacrol and l-carvone), phenylpropanoids (cinnamaldehyde and eugenol), flavonoid quercetin and ellagitannins; (ii) non-specific, by affecting the QS-related intracellular regulatory pathways by lowering regulatory small RNA expression (sulphur-containing compounds ajoene and iberin) or c-di-GMP metabolism reduction (coumarin); and (iii) indirect, via alteration of metabolic pathways involved in QS-dependent processes (vanillic acid and curcumin).

RpfC regulates the expression of the key regulator hrpX of the hrp/T3SS system in Xanthomonas campestris pv. campestris

BackgroundThe Gram-negative phytopathogenic bacterium Xanthomonas campestris pv. campestris recruits the hrp/T3SS system to inject pathogenicity effector proteins into host cells and uses the rpf/DSF cell-cell signaling system to regulate the expression of virulence factors such as extracellular enzymes and polysaccharide. Whether these two systems have any connection is unknown.MethodsPositive regulator candidates affecting hrpX expression were identified by sacB strategy. The transcriptional expression was determined by qRT-PCR and GUS activity analysis. Transcriptome analysis was performed by RNA deep-sequencing. The hypersensitive response (HR) was determined in the nonhost plant pepper ECW-10R and electrolyte leakage assay.ResultsMutation of the gene encoding the sensor RpfC of the rpf/DSF system significantly reduced the expression of hrpX, the key regulator of the hrp/T3SS system, all of the genes in the hrp cluster and most reported type III effector genes. Mutation of rpfG did not affect the expression of hrpX. The rpfC mutant showed a delayed and weakened HR induction.ConclusionsRpfC positively regulates the expression of hrpX independent of RpfG, showing a complex regulatory network linking the rpf/DSF and hrp/T3SS systems.

Photoinactivation of Pseudomonas syringae pv. actinidiae in kiwifruit plants by cationic porphyrins

The studied cationic porphyrins formulation allows an effective photoinactivation of Pseudomonas syringae pv. actinidiae in kiwifruit leaves under sunlight irradiation, without damaging the plant. Pseudomonas syringae pv. actinidiae (Psa) is a Gram-negative phytopathogenic bacterium responsible for canker on kiwifruit plant. Over the last decade, this bacterium dramatically affected the production of this fruit worldwide, causing significant economic losses. In general, Psa control consists in the application of copper which are toxic and persist in the environment. The application of antimicrobial photodynamic therapy (aPDT) as an alternative to inactivate Psa has already been demonstrated in recent studies that showed a 4 log Psa reduction using the cationic porphyrin Tetra-Py+-Me as photosensitizer (PS) and 3 consecutive cycles of treatment with a light irradiance of 150 mWcm-2. The present work aimed to evaluate the photodynamic efficiency of a new formulation constituted with five cationic porphyrins as PS in Psa inactivation. This new formulation was prepared to have as main component the tri-cationic porphyrin which is considered one of the most efficient photosensitizers in the photoinactivation of microorganisms. The in vitro study with a PS concentration of 5.0µM and low irradiance, showed a 7.4 log photoinactivation after 60min. Posteriorly, several assays were performed with the PS at 50µM on kiwifruit leaves (ex vivo), under different conditions of light and inoculation. The ex vivo assays with artificially contaminated leaves showed a 2.8 and 4.5 log inactivation with low irradiance and sunlight, respectively, after 90min. After a second treatment with sunlight, a 6.2 log inactivation was achieved. The photoinactivation on naturally contaminated leaves was about 2.3 log after 90min sunlight irradiation. Ten consecutive cycles of phototreatment in sub-lethal conditions showed that Psa does not develop resistance, nor recover viability. The results suggest that aPDT can be an alternative to the current methods used to control Psa, since it was possible to inactivate this bacterium under sunlight, without damaging the leaves.

Retraction: Detection and Functional Characterization of a 215 Amino Acid N-Terminal Extension in the Xanthomonas Type III Effector XopD.

During evolution, pathogens have developed a variety of strategies to suppress plant-triggered immunity and promote successful infection. In Gram-negative phytopathogenic bacteria, the so-called type III protein secretion system works as a molecular syringe to inject type III effectors (T3Es) into plant cells. The XopD T3E from the strain 85-10 of Xanthomonas campestris pathovar vesicatoria (Xcv) delays the onset of symptom development and alters basal defence responses to promote pathogen growth in infected tomato leaves. XopD was previously described as a modular protein that contains (i) an N-terminal DNA-binding domain (DBD), (ii) two tandemly repeated EAR (ERF-associated amphiphillic repression) motifs involved in transcriptional repression, and (iii) a C-terminal cysteine protease domain, involved in release of SUMO (small ubiquitin-like modifier) from SUMO-modified proteins. Here, we show that the XopD protein that is produced and secreted by Xcv presents an additional N-terminal extension of 215 amino acids. Closer analysis of this newly identified N-terminal domain shows a low complexity region rich in lysine, alanine and glutamic acid residues (KAE-rich) with high propensity to form coiled-coil structures that confers to XopD the ability to form dimers when expressed in E. coli. The full length XopD protein identified in this study (XopD1-760) displays stronger repression of the XopD plant target promoter PR1, as compared to the XopD version annotated in the public databases (XopD216-760). Furthermore, the N-terminal extension of XopD, which is absent in XopD216-760, is essential for XopD type III-dependent secretion and, therefore, for complementation of an Xcv mutant strain deleted from XopD in its ability to delay symptom development in tomato susceptible cultivars. The identification of the complete sequence of XopD opens new perspectives for future studies on the XopD protein and its virulence-associated functions in planta.

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.

Global Analysis of Type Three Secretion System and Quorum Sensing Inhibition of Pseudomonas savastanoi by Polyphenols Extracts from Vegetable Residues

Protection of plants against bacterial diseases still mainly relies on the use of chemical pesticides, which in Europe correspond essentially to copper-based compounds. However, recently plant diseases control is oriented towards a rational use of molecules and extracts, generally with natural origin, with lower intrinsic toxicity and a reduced negative environmental impact. In this work, polyphenolic extracts from vegetable no food/feed residues of typical Mediterranean crops, as Olea europaea, Cynara scolymus, and Vitis vinifera were obtained and their inhibitory activity on the Type Three Secretion System (TTSS) and the Quorum Sensing (QS) of the Gram-negative phytopathogenic bacterium Pseudomonas savastanoi pv. nerii strain Psn23 was assessed. Extract from green tea (Camellia sinensis) was used as a positive control. Collectively, the data obtained through gfp-promoter fusion system and real-time PCR show that all the polyphenolic extracts here studied have a high inhibitory activity on both the TTSS and QS of Psn23, without any depressing effect on bacterial viability. Extracts from green tea and grape seeds were shown to be the most active. Such activity was confirmed in planta by a strong reduction in the ability of Psn23 to develop hyperplastic galls on explants from adult oleander plants, as well as to elicit hypersensitive response on tobacco. By using a newly developed Congo red assay and an ELISA test, we demonstrated that the TTSS-targeted activity of these polyphenolic extracts also affects the TTSS pilus assembly. In consideration of the potential application of polyphenolic extracts in plant protection, the absence of any toxicity of these polyphenolic compounds was also assessed. A widely and evolutionary conserved molecular target such as Ca2+-ATPase, essential for the survival of any living organism, was used for the toxicity assessment.

Xanthomonas campestris FabH is required for branched-chain fatty acid and DSF-family quorum sensing signal biosynthesis

Xanthomonas campestris pv. campestris (Xcc), a Gram-negative phytopathogenic bacterium, causes black rot disease of cruciferous vegetables. Although Xcc has a complex fatty acid profile comprised of straight-chain fatty acids and branched-chain fatty acids (BCFAs), and encodes a complete set of genes required for fatty acid synthesis, there is still little known about the mechanism of BCFA synthesis. We reported that expression of Xcc fabH restores the growth of Ralstonia solanacearum fabH mutant, and this allows the R. solanacearum fabH mutant to produce BCFAs. Using in vitro assays, we demonstrated that Xcc FabH is able to condense branched-chain acyl-CoAs with malonyl-ACP to initiate BCFA synthesis. Moreover, although the fabH gene is essential for growth of Xcc, it can be replaced with Escherichia coli fabH, and Xcc mutants failed to produce BCFAs. These results suggest that Xcc does not have an obligatory requirement for BCFAs. Furthermore, Xcc mutants lost the ability to produce cis-11-methyl-2-dodecenoic acid, a diffusible signal factor (DSF) required for quorum sensing of Xcc, which confirms that the fatty acid synthetic pathway supplies the intermediates for DSF signal biosynthesis. Our study also showed that replacing Xcc fabH with E. coli fabH affected Xcc pathogenesis in host plants.

Mutations improving production and secretion of extracellular lipase by Burkholderia glumae PG1.

Burkholderia glumae is a Gram-negative phytopathogenic bacterium known as the causative agent of rice panicle blight. Strain B. glumae PG1 is used for the production of a biotechnologically relevant lipase, which is secreted into the culture supernatant via a type II secretion pathway. We have comparatively analyzed the genome sequences of B. glumae PG1 wild type and a lipase overproducing strain obtained by classical strain mutagenesis. Among a total number of 72 single nucleotide polymorphisms (SNPs) identified in the genome of the production strain, two were localized in front of the lipAB operon and were analyzed in detail. Both mutations contribute to a 100-fold overproduction of extracellular lipase in B. glumae PG1 by affecting transcription of the lipAB operon and efficiency of lipase secretion. We analyzed each of the two SNPs separately and observed a stronger influence of the promoter mutation than of the signal peptide modification but also a cumulative effect of both mutations. Furthermore, fusion of the mutated LipA signal peptide resulted in a 2-fold increase in secretion of the heterologous reporter alkaline phosphatase from Escherichia coli.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-015-7041-z) contains supplementary material, which is available to authorized users.

Glycan moiety of flagellin in Acidovorax avenae K1 prevents the recognition by rice that causes the induction of immune responses

Recognition of pathogen-associated molecular patterns (PAMPs) such as flagellin, a main component of the bacterial flagellum, constitutes the first layer of plant immunity and is referred to as PAMP-triggered immunity (PTI). The rice avirulent N1141 strain of gram-negative phytopathogenic bacterium, Acidovorax avenae, induces PTI including H2O2 generation, while flagellin from the rice virulent K1 strain of A. avenae does not induce these immune responses. Mass spectrometry analyses revealed that total 1,600-Da and 2,150-Da of glycan residues were present on the flagellins from N1141 and K1, respectively. A deglycosylated K1 flagellin induced immune responses in the same manner as N1141 flagellin, suggesting that the glycan in K1 flagellin prevent epitope recognition in rice. We identified three genes in K1 flagella operon, which regulate structural modification of glycan in K1 flagellin. The immature glycan-attached flagellin from three genes deletion mutant, KΔ3FG, induced H2O2 generation in cultured rice cells, whereas the K1 mature-type flagellin did not cause a detectable increase in H2O2. The data indicate that the immature glycan of flagellin from KΔ3FG cannot prevent the epitope recognition in rice.

HrcT Is a Key Component of the Type III Secretion System in Xanthomonas spp. and Also Regulates the Expression of the Key hrp Transcriptional Activator HrpX

The type III secretion system (T3SS), encoded by hrp (hypersensitive response and pathogenicity) genes in Gram-negative phytopathogenic bacteria, delivers repertoires of T3SS effectors (T3SEs) into plant cells to trigger the hypersensitive response (HR) in nonhost or resistant-host plants and promote pathogenicity in susceptible plants. The expression of hrp genes in Xanthomonas is regulated by two key regulatory proteins, HrpG and HrpX. However, the interactions between hrp gene products in directing T3SE secretion are largely unknown. Here we demonstrated that HrcT of X. oryzae pv. oryzicola functions as a T3SS component and positively regulates the expression of hrpX. Transcription of hrcT occurs via two distinct promoters; one (T1) is with the hrpB operon and the second (T3) within hrpB7 Via either promoter T1 or T3, the defect in Hrp phenotype by hrcT deletion was corrected in the presence of hrcT only from Xanthomonas species but not from other phytopathogenic bacteria. An N-terminally truncated HrcT was able to bind the hrpX promoter and activate the expression of hrpX, supporting that HrcT is a positive regulator of hrpX. A revised model showing the regulatory interactions between HrcT, HrpX, and HrpG is proposed.

Leucine zipper-like motifs of HrpZPss are not essential to induce hypersensitive response in tobacco.

Harpins are heat-stable, glycine-rich proteins secreted by several Gram-negative phytopathogenic bacteria, involved in the induction of hypersensitive response in non-host plants. HrpZPss is highly heat stable, exists as a polydispersed, multimeric protein in nature and the functional significance for its existence in many oligomeric forms, remains still unclear. It was suggested that the leucine zipper-like motifs (LZMs) may take part in the formation of oligomeric aggregates, which may be responsible for HR elicitation by harpins and for their high thermal stability. To test the involvement of LZMs in HR elicitation and induction of defense responses, we have generated deletion mutants with or without LZMs and analysed their ability to induce HR, and/or activate the defense responses. The deletion mutant HrpZMM1, with no LZM also elicited HR as well as defense responses in tobacco suggesting the LZMs are not essential for elicitor activity of HrpZPss. However, the ability to elicit defense responses may be linked with the ability to induce HR.