Erwinia amylovora Type III Secretion System Inhibitors Reduce Fire Blight Infection Under Field Conditions.

Fire blight, caused by Erwinia amylovora, is an economically important disease in apples and pears worldwide. This pathogen relies on the type III secretion system (T3SS) to cause disease. Compounds that inhibit the function of the T3SS (T3SS inhibitors) have emerged as alternative strategies for bacterial plant disease management, as they block bacterial virulence without affecting growth, unlike traditional antibiotics. In this study, we investigated the mode of action of a T3SS inhibitor named TS108, a plant phenolic acid derivative, in E. amylovora. We showed that adding TS108 to an in vitro culture of E. amylovora repressed the expression of several T3SS regulon genes, including the master regulator gene hrpL. Further studies demonstrated that TS108 negatively regulates CsrB, a global regulatory small RNA, at the posttranscriptional level, resulting in a repression of hrpS, which encodes a key activator of hrpL. Additionally, TS108 has no impact on the expression of T3SS in Dickeya dadantii or Pseudomonas aeruginosa, suggesting that its inhibition of the E. amylovora T3SS is likely species specific. To better evaluate the performance of T3SS inhibitors in fire blight management, we conducted five independent field experiments in four states (Michigan, New York, Oregon, and Connecticut) from 2015 to 2022 and observed reductions in blossom blight incidence as high as 96.7% compared with untreated trees. In summary, the T3SS inhibitors exhibited good efficacy against fire blight.

A glycine zipper motif is required for the translocation of a T6SS toxic effector into target cells.

Type VI secretion systems (T6SSs) can deliver diverse toxic effectors into eukaryotic and bacterial cells. Although much is known about the regulation and assembly of T6SS, the translocation mechanism of effectors into the periplasm and/or cytoplasm of target cells remains elusive. Here, we use the Agrobacterium tumefaciens DNase effector Tde1 to unravel the mechanism of translocation from attacker to prey. We demonstrate that Tde1 binds to its adaptor Tap1 through the N-terminus, which harbors continuous copies of GxxxG motifs resembling the glycine zipper structure found in proteins involved in the membrane channel formation. Amino acid substitutions on G39 xxxG43 motif do not affect Tde1-Tap1 interaction and secretion but abolish its membrane permeability and translocation of its fluorescent fusion protein into prey cells. The data suggest that G39 xxxG43 governs the delivery of Tde1 into target cells by permeabilizing the cytoplasmic membrane. Considering the widespread presence of GxxxG motifs in bacterial effectors and pore-forming toxins, we propose that glycine zipper-mediated permeabilization is a conserved mechanism used by bacterial effectors for translocation across target cell membranes.

The phytopathogen Dickeya dadantii 3937 cpxR locus gene participates in the regulation of virulence and the global c-di-GMP network.

Bacteria use signal transduction systems to sense and respond to their external environment. The two-component system CpxA/CpxR senses misfolded envelope protein stress and responds by up-regulating envelope protein factors and down-regulating virulence factors in several animal pathogens. Dickeya dadantii is a phytopathogen equipped with a type III secretion system (T3SS) for manipulating the host immune response. We found that deletion of cpxR enhanced the expression of the T3SS marker gene hrpA in a designated T3SS-inducing minimal medium (MM). In the ∆cpxR mutant, multiple T3SS and c-di-GMP regulators were also up-regulated. Subsequent analysis revealed that deletion of the phosphodiesterase gene egcpB in ∆cpxR abolished the enhanced T3SS expression. This suggested that CpxR suppresses EGcpB levels, causing low T3SS expression in MM. Furthermore, we found that the ∆cpxR mutant displayed low c-di-GMP phenotypes in biofilm formation and swimming. Increased production of cellular c-di-GMP by in trans expression of the diguanylate cyclase gene gcpA was negated in the ∆cpxR mutant. Here, we propose that CpxA/CpxR regulates T3SS expression by manipulating the c-di-GMP network, in turn modifying the multiple physiological activities involved in the response to environmental stresses in D. dadantii.

Quorum-Sensing Master Regulator VfmE Is a c-di-GMP Effector That Controls Pectate Lyase Production in the Phytopathogen Dickeya dadantii.

Dickeya dadantii is a phytopathogenic bacterium that causes diseases on a wide range of host plants. The pathogen secretes pectate lyases (Pel) through the type II secretion system (T2SS) that degrades the cell wall in host plants. The virulence of D. dadantii is controlled by the second messenger cyclic diguanylate monophosphate (c-di-GMP), and the homeostasis of c-di-GMP is maintained by a number of diguanylate cyclases and phosphodiesterases. Deletion of a phosphodiesterase ecpC repressed pelD transcription, and such repression can be suppressed by an additional deletion in vfmE. VfmE is an AraC type of transcriptional regulator in the Vfm quorum-sensing system. Our results suggest that VfmE is a c-di-GMP effector that functions as an activator of pel at low c-di-GMP concentrations and a repressor of pel at high c-di-GMP concentrations through regulation of the transcriptional activator SlyA. Multiple sequence alignment with known c-di-GMP effectors identified an RWIWR motif in VfmE that we demonstrate is required for the c-di-GMP binding. Mutation of R93D in the RxxxR motif eliminates the c-di-GMP-related phenotypes in Pel activity. Our results show that VfmE is not only a quorum-sensing regulator but also a c-di-GMP effector, suggesting that D. dadantii integrates the c-di-GMP signaling network with the Vfm quorum-sensing pathway during environmental adaptation. IMPORTANCE How bacteria integrate environmental cues from multiple sources to appropriately regulate adaptive phenotypes is a central question in microbiology. In Dickeya dadantii, the quorum-sensing regulator VfmE controls the key virulence factor pectate lyase (Pel). Here, we demonstrate that VfmE also binds to c-di-GMP, resulting in VfmE functioning as an activator of pel at low c-di-GMP concentrations and repressor of pel at high c-di-GMP concentrations. The RWIWR motif in VfmE is required for c-di-GMP binding, and mutation of the motif in the mutant R93D eliminates the c-di-GMP-related phenotypes in Pel activity. We propose that VfmE is an important mediator to integrate quorum-sensing signals with c-di-GMP to collectively regulate D. dadantii pathogenesis.

Agrobacterium tumefaciens Deploys a Versatile Antibacterial Strategy To Increase Its Competitiveness.

The type VI secretion system (T6SS) is a widespread antibacterial weapon capable of secreting multiple effectors for inhibition of competitor cells. Most of the effectors in the system share the same purpose of target intoxication, but the rationale for maintaining various types of effectors in a species is not well studied. In this study, we showed that a peptidoglycan amidase effector in Agrobacterium tumefaciens, Tae, cleaves d-Ala-meso-diaminopimelic acid (mDAP) and d-Glu bonds in peptidoglycan and is able to suppress the growth of Escherichia coli recipient cells. The growth suppression was effective only under the condition in which E. coli cells are actively growing. In contrast, the Tde DNase effectors in the strain possessed a dominant killing effect under carbon starvation. Microscopic analysis showed that Tde triggers cell elongation and DNA degradation, while Tae causes cell enlargement without DNA damage in E. coli recipient cells. In a rich medium, A. tumefaciens harboring only functional Tae was able to maintain competitiveness among E. coli and its own sibling cells. Growth suppression and the competitive advantage of A. tumefaciens were abrogated when recipient cells produced the Tae-specific immunity protein Tai. Given that Tae is highly conserved among A. tumefaciens strains, the combination of Tae and Tde effectors could allow A. tumefaciens to better compete with various competitors by increasing its survival during changing environmental conditions.IMPORTANCE The T6SS encodes multiple effectors with diverse functions, but little is known about the biological significance of harboring such a repertoire of effectors. We reported that the T6SS antibacterial activity of the plant pathogen Agrobacterium tumefaciens can be enhanced under carbon starvation or when recipient cell wall peptidoglycan is disturbed. This led to a newly discovered role for the T6SS peptidoglycan amidase Tae effector in providing a growth advantage dependent on the growth status of the target cell. This is in contrast to the Tde DNase effectors that are dominant during carbon starvation. Our study suggests that combining Tae and other effectors could allow A. tumefaciens to increase its competitiveness among changing environmental conditions.

Innovation and Application of the Type III Secretion System Inhibitors in Plant Pathogenic Bacteria.

Many Gram-negative pathogenic bacteria rely on a functional type III secretion system (T3SS), which injects multiple effector proteins into eukaryotic host cells, for their pathogenicity. Genetic studies conducted in different host-microbe pathosystems often revealed a sophisticated regulatory mechanism of their T3SSs, suggesting that the expression of T3SS is tightly controlled and constantly monitored by bacteria in response to the ever-changing host environment. Therefore, it is critical to understand the regulation of T3SS in pathogenic bacteria for successful disease management. This review focuses on a model plant pathogen, Dickeyadadantii, and summarizes the current knowledge of its T3SS regulation. We highlight the roles of several T3SS regulators that were recently discovered, including the transcriptional regulators: FlhDC, RpoS, and SlyA; the post-transcriptional regulators: PNPase, Hfq with its dependent sRNA ArcZ, and the RsmA/B system; and the bacterial second messenger cyclic-di-GMP (c-di-GMP). Homologs of these regulatory components have also been characterized in almost all major bacterial plant pathogens like Erwiniaamylovora, Pseudomonassyringae, Pectobacterium spp., Xanthomonas spp., and Ralstonia spp. The second half of this review shifts focus to an in-depth discussion of the innovation and development of T3SS inhibitors, small molecules that inhibit T3SSs, in the field of plant pathology. This includes T3SS inhibitors that are derived from plant phenolic compounds, plant coumarins, and salicylidene acylhydrazides. We also discuss their modes of action in bacteria and application for controlling plant diseases.

Cyclic di-GMP inactivates T6SS and T4SS activity in Agrobacterium tumefaciens.

The Type VI secretion system (T6SS) is a bacterial nanomachine that delivers effector proteins into prokaryotic and eukaryotic preys. This secretion system has emerged as a key player in regulating the microbial diversity in a population. In the plant pathogen Agrobacterium tumefaciens, the signalling cascades regulating the activity of this secretion system are poorly understood. Here, we outline how the universal eubacterial second messenger cyclic di-GMP impacts the production of T6SS toxins and T6SS structural components. We demonstrate that this has a significant impact on the ability of the phytopathogen to compete with other bacterial species in vitro and in planta. Our results suggest that, as opposed to other bacteria, c-di-GMP turns down the T6SS in A. tumefaciens thus impacting its ability to compete with other bacterial species within the rhizosphere. We also demonstrate that elevated levels of c-di-GMP within the cell decrease the activity of the Type IV secretion system (T4SS) and subsequently the capacity of A. tumefaciens to transform plant cells. We propose that such peculiar control reflects on c-di-GMP being a key second messenger that silences energy-costing systems during early colonization phase and biofilm formation, while low c-di-GMP levels unleash T6SS and T4SS to advance plant colonization.

A High-Throughput Interbacterial Competition Screen Identifies ClpAP in Enhancing Recipient Susceptibility to Type VI Secretion System-Mediated Attack by Agrobacterium tumefaciens.

The type VI secretion system (T6SS) is an effector delivery system used by Gram-negative bacteria to kill other bacteria or eukaryotic hosts to gain fitness. The plant pathogen Agrobacterium tumefaciens utilizes its T6SS to kill other bacteria, such as Escherichia coli. We observed that the A. tumefaciens T6SS-dependent killing outcome differs when using different T6SS-lacking, K-12 E. coli strains as a recipient cell. Thus, we hypothesized that the A. tumefaciens T6SS killing outcome not only relies on the T6SS activity of the attacker cells but also depends on the recipient cells. Here, we developed a high-throughput interbacterial competition platform to test the hypothesis by screening for mutants with reduced killing outcomes caused by A. tumefaciens strain C58. Among the 3,909 strains in the E. coli Keio library screened, 16 mutants with less susceptibility to A. tumefaciens C58 T6SS-dependent killing were identified, and four of them were validated by complementation test. Among the four, the clpP encoding ClpP protease, which is universal and highly conserved in both prokaryotes and eukaryotic organelles, was selected for further characterizations. We demonstrated that ClpP is responsible for enhancing susceptibility to the T6SS killing. Because ClpP protease depends on other adapter proteins such as ClpA and ClpX for substrate recognition, further mutant studies followed by complementation tests were carried out to reveal that ClpP-associated AAA+ ATPase ClpA, but not ClpX, is involved in enhancing susceptibility to A. tumefaciens T6SS killing. Moreover, functional and biochemical studies of various ClpP amino acid substitution variants provided evidence that ClpA-ClpP interaction is critical in enhancing susceptibility to the T6SS killing. This study highlights the importance of recipient factors in determining the outcome of the T6SS killing and shows the universal ClpP protease as a novel recipient factor hijacked by the T6SS of A. tumefaciens.

Stable pH Suppresses Defense Signaling and is the Key to Enhance Agrobacterium-Mediated Transient Expression in Arabidopsis Seedlings.

Agrobacterium-mediated transient expression is a powerful analysis platform for diverse plant gene functional studies, but the mechanisms regulating the expression or transformation levels are poorly studied. Previously, we developed a highly efficient and robust Agrobacterium-mediated transient expression system, named AGROBEST, for Arabidopsis seedlings. In this study, we found that AGROBEST could promote the growth of agrobacteria as well as inhibit the host immunity response. When the factor of agrobacterial growth is minimized, maintaining pH at 5.5 with MES buffer was the key to achieving optimal transient expression efficiency. The expression of plant immunity marker genes, FRK1 and NHL10, was suppressed in the pH-buffered medium as compared with non-buffered conditions in Col-0 and an efr-1 mutant lacking the immunity receptor EFR recognizing EF-Tu, a potent pathogen- or microbe-associated molecular pattern (PAMP or MAMP) of A. tumefaciens. Notably, such immune suppression could also occur in Arabidopsis seedlings without Agrobacterium infection. Furthermore, the PAMP-triggered influx of calcium ions was compromised in the pH-buffered medium. We propose that the enhanced transient expression efficiency by stable pH was due to inhibiting calcium ion uptake and subsequently led to suppressing immunity against Agrobacterium.

Redox regulation of pyruvate kinase M2 by cysteine oxidation and S-nitrosation.

We show here that the M2 isoform of human pyruvate kinase (M2PYK) is susceptible to nitrosation and oxidation, and that these modifications regulate enzyme activity by preventing the formation of the active tetrameric form. The biotin-switch assay carried out on M1 and M2 isoforms showed that M2PYK is sensitive to nitrosation and that Cys326 is highly susceptible to redox modification. Structural and enzymatic studies have been carried out on point mutants for three cysteine residues (Cys424, Cys358, and Cys326) to characterise their potential roles in redox regulation. Nine cysteines are conserved between M2PYK and M1PYK. Cys424 is the only cysteine unique to M2PYK. C424S, C424A, and C424L showed a moderate effect on enzyme activity with 80, 100, and 140% activity, respectively, compared with M2PYK. C358 had been previously identified from in vivo studies to be the favoured target for oxidation. Our characterised mutant showed that this mutation stabilises tetrameric M2PYK, suggesting that the in vivo resistance to oxidation for the Cys358Ser mutation is due to stabilisation of the tetrameric form of the enzyme. In contrast, the Cys326Ser mutant exists predominantly in monomeric form. A biotin-switch assay using this mutant also showed a significant reduction in biotinylation of M2PYK, confirming that this is a major target for nitrosation and probably oxidation. Our results show that the sensitivity of M2PYK to oxidation and nitrosation is regulated by its monomer-tetramer equilibrium. In the monomer state, residues (in particular C326) are exposed to oxidative modifications that prevent reformation of the active tetrameric form.

Functional Exploration of the Bacterial Type VI Secretion System in Mutualism: Azorhizobium caulinodans ORS571-Sesbania rostrata as a Research Model.

The bacterial type VI secretion system (T6SS) has been considered the armed force of bacteria because it can deliver toxin effectors to prokaryotic or eukaryotic cells for survival and fitness. Although many legume symbiotic rhizobacteria encode T6SS in their genome, the biological function of T6SS in these bacteria is still unclear. To elucidate this issue, we used Azorhizobium caulinodans ORS571 and its symbiotic host Sesbania rostrata as our research model. By using T6SS gene deletion mutants, we found that T6SS provides A. caulinodans with better symbiotic competitiveness when coinfected with a T6SS-lacking strain, as demonstrated by two independent T6SS-deficient mutants. Meanwhile, the symbiotic effectiveness was not affected by T6SS because the nodule phenotype, nodule size, and nodule nitrogen-fixation ability did not differ between the T6SS mutants and the wild type when infected alone. Our data also suggest that under several lab culture conditions tested, A. caulinodans showed no T6SS-dependent interbacterial competition activity. Therefore, instead of being an antihost or antibacterial weapon of the bacterium, the T6SS in A. caulinodans ORS571 seems to participate specifically in symbiosis by increasing its symbiotic competitiveness.

Differentially localized rice ethylene receptors OsERS1 and OsETR2 and their potential role during submergence.

Ethylene is gaseous plant hormone that controls a variety of physiologic activities. OsERS1 and OsETR2 are major ethylene receptors in rice that have been reported to have different regulatory functions. The GFP fused N-terminus of OsERS1 and OsETR2 showed differentially localization patterns when transiently expressed in onion epidermal cells. Base on these results, we suggested that OsERS1 could be localized to plasma membranes, whereas OsETR2 could be localized to the endoplasmic reticulum. Furthermore, instead of the constitutive expression profile of OsERS1, OsETR2 is differentially expressed in seedlings of light/dark-grown conditions, submergence or exogenous ethylene treatments. Our results and others support the notion that OsERS1 and OsETR2 could have different roles during rice plant submergence.

Warfare between Host Immunity and Bacterial Weapons.

Bacterial pathogens deploy protein secretion systems to facilitate infection and colonization of their hosts. In this issue of Cell Host & Microbe, Chen et al. (2017) report a new role for a type VI secretion effector in promoting bacterial colonization by preventing inflammasome activation induced by a type III secretion system.

Agrobacterium-mediated plant transformation: biology and applications.

Plant genetic transformation heavily relies on the bacterial pathogen Agrobacterium tumefaciens as a powerful tool to deliver genes of interest into a host plant. Inside the plant nucleus, the transferred DNA is capable of integrating into the plant genome for inheritance to the next generation (i.e. stable transformation). Alternatively, the foreign DNA can transiently remain in the nucleus without integrating into the genome but still be transcribed to produce desirable gene products (i.e. transient transformation). From the discovery of A. tumefaciens to its wide application in plant biotechnology, numerous aspects of the interaction between A. tumefaciens and plants have been elucidated. This article aims to provide a comprehensive review of the biology and the applications of Agrobacterium-mediated plant transformation, which may be useful for both microbiologists and plant biologists who desire a better understanding of plant transformation, protein expression in plants, and plant-microbe interaction.

Identification of S-Nitrosothiols by the Sequential Cysteine Blocking Technique.

Here, we describe a procedure for the identification of S-nitrosothiols that has been used in our laboratory to study the roles of protein S-nitrosylation in the immune responses of Arabidopsis thaliana and other organisms. It employs a modified version of the biotin-switch technique, which we termed the sequential cysteine blocking technique, encompassing the sequential redox-blocking of recombinant proteins followed by LC-MS/MS analysis.

Nitric oxide and S-nitrosoglutathione function additively during plant immunity.

Nitric oxide (NO) is emerging as a key regulator of diverse plant cellular processes. A major route for the transfer of NO bioactivity is S-nitrosylation, the addition of an NO moiety to a protein cysteine thiol forming an S-nitrosothiol (SNO). Total cellular levels of protein S-nitrosylation are controlled predominantly by S-nitrosoglutathione reductase 1 (GSNOR1) which turns over the natural NO donor, S-nitrosoglutathione (GSNO). In the absence of GSNOR1 function, GSNO accumulates, leading to dysregulation of total cellular S-nitrosylation. Here we show that endogenous NO accumulation in Arabidopsis, resulting from loss-of-function mutations in NO Overexpression 1 (NOX1), led to disabled Resistance (R) gene-mediated protection, basal resistance and defence against nonadapted pathogens. In nox1 plants both salicylic acid (SA) synthesis and signalling were suppressed, reducing SA-dependent defence gene expression. Significantly, expression of a GSNOR1 transgene complemented the SNO-dependent phenotypes of paraquat resistant 2-1 (par2-1) plants but not the NO-related characters of the nox1-1 line. Furthermore, atgsnor1-3 nox1-1 double mutants supported greater bacterial titres than either of the corresponding single mutants. Our findings imply that GSNO and NO, two pivotal redox signalling molecules, exhibit additive functions and, by extension, may have distinct or overlapping molecular targets during both immunity and development.

Nitric oxide function in plant biology: a redox cue in deconvolution.

Nitric oxide (NO), a gaseous, redox-active small molecule, is gradually becoming established as a central regulator of growth, development, immunity and environmental interactions in plants. A major route for the transfer of NO bioactivity is S-nitrosylation, the covalent attachment of an NO moiety to a protein cysteine thiol to form an S-nitrosothiol (SNO). This chemical transformation is rapidly emerging as a prototypic, redox-based post-translational modification integral to the life of plants. Here we review the myriad roles of NO and SNOs in plant biology and, where known, the molecular mechanisms underpining their activity.

A sleigh ride through the SNO: regulation of plant immune function by protein S-nitrosylation.

S-nitrosylation, the covalent attachment of a nitric oxide (NO) moiety to a protein cysteine thiol to form an S-nitrosothiol (SNO) is rapidly emerging as a prototypic, redox-based post-translational modification during plant immune function. Here we review recently identified targets for S-nitrosylation and the consequences of these modifications in relation to the control of plant disease resistance.

S-nitrosylation of NADPH oxidase regulates cell death in plant immunity.

Changes in redox status are a conspicuous feature of immune responses in a variety of eukaryotes, but the associated signalling mechanisms are not well understood. In plants, attempted microbial infection triggers the rapid synthesis of nitric oxide and a parallel accumulation of reactive oxygen intermediates, the latter generated by NADPH oxidases related to those responsible for the pathogen-activated respiratory burst in phagocytes. Both nitric oxide and reactive oxygen intermediates have been implicated in controlling the hypersensitive response, a programmed execution of plant cells at sites of attempted infection. However, the molecular mechanisms that underpin their function and coordinate their synthesis are unknown. Here we show genetic evidence that increases in cysteine thiols modified using nitric oxide, termed S-nitrosothiols, facilitate the hypersensitive response in the absence of the cell death agonist salicylic acid and the synthesis of reactive oxygen intermediates. Surprisingly, when concentrations of S-nitrosothiols were high, nitric oxide function also governed a negative feedback loop limiting the hypersensitive response, mediated by S-nitrosylation of the NADPH oxidase, AtRBOHD, at Cys 890, abolishing its ability to synthesize reactive oxygen intermediates. Accordingly, mutation of Cys 890 compromised S-nitrosothiol-mediated control of AtRBOHD activity, perturbing the magnitude of cell death development. This cysteine is evolutionarily conserved and specifically S-nitrosylated in both human and fly NADPH oxidase, suggesting that this mechanism may govern immune responses in both plants and animals.

Topological analysis of a haloacid permease of a Burkholderia sp. bacterium with a PhoA-LacZ reporter.

BACKGROUND: 2-Haloacids can be found in the natural environment as degradative products of natural and synthetic halogenated compounds. They can also be generated by disinfection of water and have been shown to be mutagenic and to inhibit glyceraldehyde-3-phosphate dehydrogenase activity. We have recently identified a novel haloacid permease Deh4p from a bromoacetate-degrading bacterium Burkholderia sp. MBA4. Comparative analyses suggested that Deh4p is a member of the Major Facilitator Superfamily (MFS), which includes thousands of membrane transporter proteins. Members of the MFS usually possess twelve putative transmembrane segments (TMS). Deh4p was predicted to have twelve TMS. In this study we characterized the topology of Deh4p with a PhoA-LacZ dual reporters system. RESULTS: Thirty-six Deh4p-reporter recombinants were constructed and expressed in E. coli. Both PhoA and LacZ activities were determined in these cells. Strength indices were calculated to determine the locations of the reporters. The results mainly agree with the predicted model. However, two of the TMS were not verified. This lack of confirmation of the TMS, using a reporter, has been reported previously. Further comparative analysis of Deh4p has assigned it to the Metabolite:H+ Symporter (MHS) 2.A.1.6 family with twelve TMS. Deh4p exhibits many common features of the MHS family proteins. Deh4p is apparently a member of the MFS but with some atypical features. CONCLUSION: The PhoA-LacZ reporter system is convenient for analysis of the topology of membrane proteins. However, due to the limitation of the biological system, verification of some of the TMS of the protein was not successful. The present study also makes use of bioinformatic analysis to verify that the haloacid permease Deh4p of Burkholderia sp. MBA4 is a MFS protein but with atypical features.