Regulation of Burkholderia cenocepacia virulence by the fatty acyl-CoA ligase DsfR as a response regulator of quorum sensing signal.

Quorum sensing (QS) is a cell-to-cell communication mechanism mediated by small diffusible signaling molecules. Previous studies showed that RpfR controls Burkholderia cenocepacia virulence as a cis-2-dodecenoic acid (BDSF) QS signal receptor. Here, we report that the fatty acyl-CoA ligase DsfR (BCAM2136), which efficiently catalyzes in vitro synthesis of lauryl-CoA and oleoyl-CoA from lauric acid and oleic acid, respectively, acts as a global transcriptional regulator to control B. cenocepacia virulence by sensing BDSF. We show that BDSF binds to DsfR with high affinity and enhances the binding of DsfR to the promoter DNA regions of target genes. Furthermore, we demonstrate that the homolog of DsfR in B. lata, RS02960, binds to the target gene promoter, and perception of BDSF enhances the binding activity of RS02960. Together, these results provide insights into the evolved unusual functions of DsfR that control bacterial virulence as a response regulator of QS signal.

Phytopathogenic bacteria utilize host glucose as a signal to stimulate virulence through LuxR homologues.

Chemical signal-mediated biological communication is common within bacteria and between bacteria and their hosts. Many plant-associated bacteria respond to unknown plant compounds to regulate bacterial gene expression. However, the nature of the plant compounds that mediate such interkingdom communication and the underlying mechanisms remain poorly characterized. Xanthomonas campestris pv. campestris (Xcc) causes black rot disease on brassica vegetables. Xcc contains an orphan LuxR regulator (XccR) which senses a plant signal that was validated to be glucose by HPLC-MS. The glucose concentration increases in apoplast fluid after Xcc infection, which is caused by the enhanced activity of plant sugar transporters translocating sugar and cell-wall invertases releasing glucose from sucrose. XccR recruits glucose, but not fructose, sucrose, glucose 6-phosphate, and UDP-glucose, to activate pip expression. Deletion of the bacterial glucose transporter gene sglT impaired pathogen virulence and pip expression. Structural prediction showed that the N-terminal domain of XccR forms an alternative pocket neighbouring the AHL-binding pocket for glucose docking. Substitution of three residues affecting structural stability abolished the ability of XccR to bind to the luxXc box in the pip promoter. Several other XccR homologues from plant-associated bacteria can also form stable complexes with glucose, indicating that glucose may function as a common signal molecule for pathogen-plant interactions. The conservation of a glucose/XccR/pip-like system in plant-associated bacteria suggests that some phytopathogens have evolved the ability to utilize host compounds as virulence signals, indicating that LuxRs mediate an interkingdom signalling circuit.

Type III Secretion System Repressor RhpR Induces GrlP, a Glycine-Rich Outer Membrane Lipoprotein with Functions in Regulating the Periplasmic Space and Pleiotropic Responses.

The two-component system RhpRS was initially identified as a regulator of genes encoding the type III secretion system (T3SS) in Pseudomonas syringae. Phosphorylated RhpR (P-RhpR) negatively regulates the T3SS genes by repressing the hrpR promoter, but directly activates the expression of a small gene named here as grlp. Here, we show that grlp is expressed higher in rich medium than in minimal medium in P. s. pv. tomato DC3000 and encodes a glycine rich lipoprotein (GrlP) located in the outer membrane (OM). The grlp gene has a pleiotropic effect on bacterial behaviors such as reductions in pathogenicity, swimming motility, biofilm formation, tolerance to various stresses and antibiotics, and long-term survival when overexpressed, but induces these responses when it is deleted in P. s. pv. tomato DC3000. Overexpression of grlp increases the size of periplasm while deletion of grlp decreases the periplasmic space. Further, GrlP interacts with OprI, the ortholog of E. coli OM lipoprotein Lpp, a key player in determining the size of periplasm and mechanic stiffness of the OM by tethering the OM to peptidoglycan (PG) in periplasm. As periplasmic space and OM mechanics play central roles in regulating bacterial physiology, we speculate that GrlP probably imposes its functions on bacterial physiology by regulating the periplasmic space and OM mechanics. These findings suggest that the T3SS gene regulation is closely coordinated with bacterial cell envelope properties by RhpRS in P. syringe. IMPORTANCE The OM of Gram-negative bacteria is the most front line in contact with extracellular milieu. OM is not only a protective layer, but also a structure that determines the envelope stiffness. Recent evidence indicated that components determining the periplasmic space and cross-links of lipopolysaccharide on the OM play key roles in regulating the mechanical properties of the OM. However, whether the OM composition and mechanical properties are coordinated with the expression of the T3SS genes is unknown. Here, we found that the two-component system (TCS) regulator P-RhpR, a direct repressor of the T3SS regulator hrpRS operon, directly activates the expression of the OM lipoprotein gene grlp bearing a function in regulating the periplasmic space. This finding suggests a coordination between the OM properties and the T3SS gene regulation and reveals a new target for control of the T3SS gene expression and bacterial pathogenicity.

N-3-Oxo-Octanoyl Homoserine Lactone Primes Plant Resistance Against Necrotrophic Pathogen Pectobacterium carotovorum by Coordinating Jasmonic Acid and Auxin-Signaling Pathways.

Many Gram-negative bacteria use small signal molecules, such as N-acyl-homoserine lactones (AHLs), to communicate with each other and coordinate their collective behaviors. Recently, increasing evidence has demonstrated that long-chained quorum-sensing signals play roles in priming defense responses in plants. Our previous work indicated that a short-chained signal, N-3-oxo-octanoyl homoserine lactone (3OC8-HSL), enhanced Arabidopsis resistance to the hemi-biotrophic bacteria Pseudomonas syringae pv. tomato DC3000 through priming the salicylic acid (SA) pathway. Here, we found that 3OC8-HSL could also prime resistance to the necrotrophic bacterium Pectobacterium carotovorum ssp. carotovorum (Pcc) through the jasmonic acid (JA) pathway, and is dependent on auxin responses, in both Chinese cabbage and Arabidopsis. The subsequent Pcc invasion triggered JA accumulation and increased the down-stream genes' expressions of JA synthesis genes (LOX, AOS, and AOC) and JA response genes (PDF1.2 and VSP2). The primed state was not observed in the Arabidopsis coi1-1 and jar1-1 mutants, which indicated that the primed resistance to Pcc was dependent on the JA pathway. The 3OC8-HSL was not transmitted from roots to leaves and it induced indoleacetic acid (IAA) accumulation and the DR5 and SAUR auxin-responsive genes' expressions in seedlings. When Arabidopsis and Chinese cabbage roots were pretreated with exogenous IAA (10 µM), the plants had activated the JA pathway and enhanced resistance to Pcc, which implied that the JA pathway was involved in AHL priming by coordinating with the auxin pathway. Our findings provide a new strategy for the prevention and control of soft rot in Chinese cabbage and provide theoretical support for the use of the quorum-sensing AHL signal molecule as a new elicitor.

RIR1 represses plant immunity by interacting with mitochondrial complex I subunit in rice.

We previously observed decreased expression of rice OsmiR159a.1 on infection with the bacterial blight-causing pathogen Xanthomonas oryzae pv. oryzae (Xoo), and identified the OsLRR_RLK (leucine-rich repeat_ receptor like kinase) gene as an authentic target of OsmiR159a.1. Here, we found that a Tos17 insertion mutant of LRR_RLK displayed increasing temporal resistance to Xoo, whereas the LRR_RLK overexpression lines were susceptible to the pathogen early on in the infection, indicating that LRR_RLK encodes a repressor of rice resistance to Xoo infection, and it was renamed as RIR1 (Rice Immunity Repressor 1). RIR1 overexpression plants were more susceptible to Xoo at late growth stage, suggesting that RIR1 mRNA levels are negatively correlated with the resistance of rice against Xoo. We discovered that OsmiR159a.1 repression in Xoo-infected plants was largely dependent on the pathogen's type III secretion system. Co-immunoprecipitation, bimolecular fluoresence complementation, and pull-down assays indicated that RIR1 interacted with the NADH-ubiquinone oxidoreductase (NUO) 51-kDa subunit of the mitochondrial complex I through its kinase domain. Notably, impairment of RIR1 or overexpression of NUO resulted in reactive oxygen species accumulation and enhanced expression of pathogen-resistance genes, including jasmonic acid pathway genes. We propose that pathogens may inhibit OsmiR159 to interfere with the RIR1-NUO interaction, and subsequently depression of rice immune signalling pathways. The resistance genes manipulated by Xoo can be a probe to explore the regulatory network during host-pathogen interactions.

Whole genome sequencing of Enterobacter mori, an emerging pathogen of kiwifruit and the potential genetic adaptation to pathogenic lifestyle.

Members of the Enterobacter genus are gram-negative bacteria, which are used as plant growth-promoting bacteria, and increasingly recovered from economic plants as emerging pathogens. A new Enterobacter mori strain, designated CX01, was isolated as an emerging bacterial pathogen of a recent outbreak of kiwifruit canker-like disease in China. The main symptoms associated with this syndrome are bleeding cankers on the trunk and branch, and brown leaf spots. The genome sequence of E. mori CX01 was determined as a single chromosome of 4,966,908 bp with 4640 predicted open reading frames (ORFs). To better understand the features of the genus and its potential pathogenic mechanisms, five available Enterobacter genomes were compared and a pan-genome of 4870 COGs with 3158 core COGs were revealed. An important feature of the E. mori CX01 genome is that it lacks a type III secretion system often found in pathogenic bacteria, instead it is equipped with type I, II, and VI secretory systems. Besides, the genes encoding putative virulence effectors, two-component systems, nutrient acquisition systems, proteins involved in phytohormone synthesis, which may contribute to the virulence and adaption to the host plant niches are included. The genome sequence of E. mori CX01 has high similarity with that of E. mori LMG 25,706, though the rearrangements occur throughout two genomes. Further pathogenicity assay showed that both strains can either invade kiwifruit or mulberry, indicating they may have similar host range. Comparison with a closely related isolate enabled us to understand its pathogenesis and ecology.

5S Multifunctional Intelligent Coating with Superdurable, Superhydrophobic, Self-Monitoring, Self-Heating, and Self-Healing Properties for Existing Construction Application.

There is a constant drive to develop ultra-high-performance multifunctional coatings for existing construction used in modern engineering technologies. For these materials to be used in unsound infrastructure protections, they are required to present enhanced robustness while bearing functionalities to meet multiple uses. Single-function coating is not smart enough to provide satisfactory protection, and the preparation process of multifunctional materials is complex, costly, and provides poor durability. Thus, existing coatings are not suitable to generate an intelligent closed-loop protection system. Herein, we report an innovative 5S multifunctional intelligent coating (5SC) for existing construction materials with superdurable, superhydrophobic, self-monitoring, self-heating, and self-healing properties. The 5SC material showed highly durable superhydrophobic properties as revealed by the main failure tests of building materials including physical friction (abrasion, scratching), 100% tensile strain, photoaging (3000 h of ultraviolet (UV) aging), acid corrosion (concentrated hydrochloric acid and sulfuric acid), and freeze-thaw aging (salty solution). The coated surface was highly sensitive to pressure, with monitoring thresholds from 1 to 30 000 N per 0.01 m2. It showed an early heating rate as high as 6 °C/min while maintaining very good self-monitoring and ice-melting drainage performance to protect the existing structures. This novel composite material is suitable for constructions in extreme areas where corrosion and freeze-thaw damage can occur. This multifunctional material presents a very broad range of applications and development potential in the construction field.

Two components of the rhpPC operon coordinately regulate the type III secretion system and bacterial fitness in Pseudomonas savastanoi pv. phaseolicola.

Many plant bacterial pathogens including Pseudomonas species, utilize the type III secretion system (T3SS) to deliver effector proteins into plant cells. Genes encoding the T3SS and its effectors are repressed in nutrient-rich media but are rapidly induced after the bacteria enter a plant or are transferred into nutrient-deficient media. To understand how the T3SS genes are regulated, we screened for P. savastanoi pv. phaseolicola (Psph) mutants displaying diminished induction of avrPto-luc, a reporter for the T3SS genes, in Arabidopsis. A mutant carrying transposon insertion into a gene coding for a small functional unknown protein, designated as rhpC, was identified that poorly induced avrPto-luc in plants and in minimal medium (MM). Interestingly, rhpC is located immediately downstream of a putative metalloprotease gene named rhpP, and the two genes are organized in an operon rhpPC; but rhpP and rhpC displayed different RNA expression patterns in nutrient-rich King's B medium (KB) and MM. Deletion of the whole rhpPC locus did not significantly affect the avrPto-luc induction, implying coordinated actions of rhpP and rhpC in regulating the T3SS genes. Further analysis showed that RhpC was a cytoplasmic protein that interacted with RhpP and targeted RhpP to the periplasm. In the absence of RhpC, RhpP was localized in the cytoplasm and caused a reduction of HrpL, a key regulator of the T3SS genes, and also reduced the fitness of Psph. Expression of RhpP alone in E. coli inhibited the bacterial growth. The detrimental effect of RhpP on the fitness of Psph and E. coli required metalloprotease active sites, and was repressed when RhpC was co-expressed with RhpP. The coordination between rhpP and rhpC in tuning the T3SS gene expression and cell fitness reveals a novel regulatory mechanism for bacterial pathogenesis. The function of RhpP in the periplasm remains to be studied.

Xanthomonas campestris Promotes Diffusible Signal Factor Biosynthesis and Pathogenicity by Utilizing Glucose and Sucrose from Host Plants.

The plant pathogen Xanthomonas campestris pv. campestris produces diffusible signal factor (DSF) quorum sensing (QS) signals to regulate its biological functions and virulence. Our previous study showed that X. campestris pv. campestris utilizes host plant metabolites to enhance the biosynthesis of DSF family signals. However, it is unclear how X. campestris pv. campestris benefits from the metabolic products of the host plant. In this study, we observed that the host plant metabolites not only boosted the production of the DSF family signals but also modulated the expression levels of DSF-regulated genes in X. campestris pv. campestris. Infection with X. campestris pv. campestris induced changes in the expression of many sugar transporter genes in Arabidopsis thaliana. Exogenous addition of sucrose or glucose, which are the major products of photosynthesis in plants, enhanced DSF signal production and X. campestris pv. campestris pathogenicity in the Arabidopsis model. In addition, several sucrose hydrolase-encoding genes in X. campestris pv. campestris and sucrose invertase-encoding genes in the host plant were notably upregulated during the infection process. These enzymes hydrolyzed sucrose to glucose and fructose, and in trans expression of one of these enzymes, CINV1 of A. thaliana or XC_0805 of X. campestris pv. campestris, enhanced DSF signal biosynthesis in X. campestris pv. campestris in the presence of sucrose. Taken together, our findings demonstrate that X. campestris pv. campestris applies multiple strategies to utilize host plant sugars to enhance QS and pathogenicity.

A dual role for proline iminopeptidase in the regulation of bacterial motility and host immunity.

During plant-pathogen interactions, pathogenic bacteria have evolved multiple strategies to cope with the sophisticated defence systems of host plants. Proline iminopeptidase (PIP) is essential to Xanthomonas campestris pv. campestris (Xcc) virulence, and is conserved in many plant-associated bacteria, but its pathogenic mechanism remains unclear. In this study, we found that disruption of pip in Xcc enhanced its flagella-mediated bacterial motility by decreasing intracellular bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) levels, whereas overexpression of pip in Xcc restricted its bacterial motility by elevating c-di-GMP levels. We also found that PIP is a type III secretion system-dependent effector capable of eliciting a hypersensitive response in non-host, but not host plants. When we transformed pip into the host plant Arabidopsis, higher bacterial titres were observed in pip-overexpressing plants relative to wild-type plants after Xcc inoculation. The repressive function of PIP on plant immunity was dependent on PIP's enzymatic activity and acted through interference with the salicylic acid (SA) biosynthetic and regulatory genes. Thus, PIP simultaneously regulates two distinct regulatory networks during plant-microbe interactions, i.e. it affects intracellular c-di-GMP levels to coordinate bacterial behaviour, such as motility, and functions as a type III effector translocated into plant cells to suppress plant immunity. Both processes provide bacteria with the regulatory potential to rapidly adapt to complex environments, to utilize limited resources for growth and survival in a cost-efficient manner and to improve the chances of bacterial survival by helping pathogens to inhabit the internal tissues of host plants.

Burkholderia cenocepacia integrates cis-2-dodecenoic acid and cyclic dimeric guanosine monophosphate signals to control virulence.

Quorum sensing (QS) signals are used by bacteria to regulate biological functions in response to cell population densities. Cyclic diguanosine monophosphate (c-di-GMP) regulates cell functions in response to diverse environmental chemical and physical signals that bacteria perceive. In Burkholderia cenocepacia, the QS signal receptor RpfR degrades intracellular c-di-GMP when it senses the QS signal cis-2-dodecenoic acid, also called Burkholderia diffusible signal factor (BDSF), as a proxy for high cell density. However, it was unclear how this resulted in control of BDSF-regulated phenotypes. Here, we found that RpfR forms a complex with a regulator named GtrR (BCAL1536) to enhance its binding to target gene promoters under circumstances where the BDSF signal binds to RpfR to stimulate its c-di-GMP phosphodiesterase activity. In the absence of BDSF, c-di-GMP binds to the RpfR-GtrR complex and inhibits its ability to control gene expression. Mutations in rpfR and gtrR had overlapping effects on both the B. cenocepacia transcriptome and BDSF-regulated phenotypes, including motility, biofilm formation, and virulence. These results show that RpfR is a QS signal receptor that also functions as a c-di-GMP sensor. This protein thus allows B. cenocepacia to integrate information about its physical and chemical surroundings as well as its population density to control diverse biological functions including virulence. This type of QS system appears to be widely distributed in beta and gamma proteobacteria.

[Function of biofilms in phytopathogenic bacterial-host interactions].

Biofilms are complex three-dimensional bacterial assemblages that attach to biotic or abiotic solid surfaces, and frequently embed within a self-produced matrix of extracellular polymeric substances. Biofilm formation is a microbial defense response to biotic and abiotic stresses, and a key factor for survival in adverse environments. A wide variety of microorganisms can colonize distant tissues of higher plants, such as leaves, vascular network and roots, and adhere to the surface of the tissues to form biofilms. The dynamic processes in forming biofilms in response to plant internal environment are key steps required for full virulence of phytopathogenic bacteria. Exploring the mechanisms involved in regulation of bacterial biofilms is important for understanding the plant-pathogens interactions. In this review, we summarized the research progresses related to the biofilms of bacterial phytopathogens, including biofilm characteristics, essential regulatory mechanisms and key signals affecting the transition between a planktonic lifestyle and multicellular behavior.

Interkingdom signaling in plant-microbe interactions.

The widespread communications between prokaryotes and eukaryotes via signaling molecules are believed to affect gene expression in both partners. During the communication process, the contacted organisms produce and release small molecules that establish communication channels between two kingdoms-this procedure is known as interkingdom signaling. Interkingdom communications are widespread between pathogenic or beneficial bacteria and their host plants, with diversified outcomes depending on the specific chemical-triggered signaling pathways. Deciphering the signals or language of this interkingdom communication and uncovering the underlying mechanisms are major current challenges in this field. It is evident that diverse signaling molecules can be produced or derived from bacteria and plants, and researchers have sought to identify these signals and explore the mechanisms of the signaling pathways. The results of such studies will lead to the development of strategies to improve plant disease resistance through controlling interkingdom signals, rather than directly killing the pathogenic bacteria. Also, the identification of signals produced by beneficial bacteria will be useful for agricultural applications. In this review, we summarize the recent progress of cross-kingdom interactions between plant and bacteria, and how LuxR-family transcription factors in plant associated bacterial quorum sensing system are involved in the interkingdom signaling.

Dynamic and Coordinated Expression Changes of Rice Small RNAs in Response to Xanthomonas oryzae pv. oryzae.

Endogenous small RNAs are newly identified players in plant immune responses, yet their roles in rice (Oryza sativa) responding to pathogens are still less understood, especially for pathogens that can cause severe yield losses. We examined the small RNA expression profiles of rice leaves at 2, 6, 12, and 24 hours post infection of Xanthomonas oryzae pv. oryzae (Xoo) virulent strain PXO99, the causal agent of rice bacterial blight disease. Dynamic expression changes of some miRNAs and trans-acting siRNAs were identified, together with a few novel miRNA targets, including an RLK gene targeted by osa-miR159a.1. Coordinated expression changes were observed among some small RNAs in response to Xoo infection, with small RNAs exhibiting the same expression pattern tended to regulate genes in the same or related signaling pathways, including auxin and GA signaling pathways, nutrition and defense-related pathways. These findings reveal the dynamic and complex roles of small RNAs in rice-Xoo interactions, and identify new targets for regulating plant responses to Xoo.

[Efficient transient expression to analyze miRNA targets in rice protoplasts].

Compared with the transgenic approach, transient assays provide a convenient alternative to analyze gene expression. To analyze the relationship between miRNAs and their target genes, a rice protoplast system to detect target gene activity was established. The MIRNA and GFP-fused target sequence (or GFP-fused mutated sequence as a non-target control) were constructed into the same plasmid, and then delivered into rice protoplasts. The GFP expression level decreased significantly when the protoplasts were transfected with the plasmid containing GFP-fused target compared to that of the plasmid with non-target sequence either by fluorescence microscopy or qRT-PCR method. Two microRNA genes, osaMIR156 and osaMIR397, and their target sequences were used to prove the feasibility of the rice protoplast transient assay system. This method will facilitate large-scale screening of rice miRNA target in vivo, and may be suitable for functional analysis of miRNAs of other monocot plants that might share the evolutionarily conserved small RNA processing system with rice.

Synergistic activation of the pathogenicity-related proline iminopeptidase gene in Xanthomonas campestris pv. campestris by HrpX and a LuxR homolog.

Xanthomonas campestris pv. campestris strain 8004 contains an orphan quorum-sensing (QS) locus, xccR-pip(Xcc), in which the proline iminopeptidase (pip(Xcc)) gene (where "Xcc" indicates that the pip gene is from X. campestris pv. campestris) is positively regulated by the LuxR homologue XccR by binding to the luxXc box of the pip(Xcc) promoter. The disruption of pip(Xcc) significantly attenuated the virulence of X. campestris pv. campestris. An imperfect plant-inducible promoter (PIP) box is located in the upstream region of the pip(Xcc) promoter, which is the putative binding site of the transcriptional activator HrpX. To explore whether the expression of the pip(Xcc) gene is regulated by HrpX, the expression level of a pip(Xcc) promoter-gusA fusion gene was assayed in an hrpX disruption mutant. The results showed that the lack of HrpX dramatically decreased the ß-glucuronidase (GUS) activity. Further analyses using an electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-PCR indicated that the imperfect PIP box in X. campestris pv. campestris is specifically bound to HrpX. These data demonstrated that the pip(Xcc) gene belongs to the hrp regulon and that the imperfect PIP box of the pip(Xcc) promoter could be a cis element for the HrpX protein. We further showed in a pulldown assay that XccR can bind HrpX, suggesting that these two regulatory proteins coactivate the virulence factor by binding to the different cis elements of the pip(Xcc) gene and adapt to the host environment during X. campestris pv. campestris infection.

[Implication of post-translational modifications in suppressor activity and stability of the Cucumber mosaic virus 2b protein].

To gain insights into the function of potential post-translational modifications on the activity of the Cucumber mosaic virus (CMV)-encoded silencing suppressor protein 2b, one predicted phosphorylation site (S40) and two predicted ubiquitination/sumoylation sites (K22 and K39) in CMV-Q2b protein were individually or simultaneously mutated by site-directed mutagenesis methods. These Q2b mutants were inserted into plant expression vectors, expressed in plant leaves, and then analyzed for their silencing suppressor activities. The results showed that S40A mutation greatly impaired both the local and systemic silencing suppressor activity, and the K22R mutation has no significant effect on the suppressor activity, while the K22R/K39R double mutation reduced the systemic silencing suppressor activity. To test if the decrease of suppressor activity were due to protein accumulation changes, western blot were performed to monitor the protein level of Q2b mutants. The results indicated that mutations of both K22 and K39 to R or S40 to A all significantly reduced the accumulation of the Q2b protein in plants, while the single mutation of K22 to R did not alter the accumulation of Q2b protein, suggesting that two potential post-translational modification sites, K39 and S40, contribute to the suppressor activity and stability of 2b protein in plant cells.

XerR, a negative regulator of XccR in Xanthomonas campestris pv. campestris, relieves its repressor function in planta.

We previously reported that XccR, a LuxR-type regulator of Xanthomonas campestris pv. campestris (Xcc), activates the downstream proline iminopeptidase virulence gene (pip) in response to certain host plant factor(s). In this report, we further show that the expression of the xccR gene was repressed in the culture medium by an NtrC-type response regulator, which we named XerR (XccR expression-related, repressor), and that this repression was relieved when the bacteria were grown in planta. Such a regulatory mechanism is reinforced by the observations that XerR directly bound to the xccR promoter in vitro, and that mutations at the phosphorylation-related residues of XerR resulted in the loss of its repressor function. Furthermore, the expression level of xccR increased even in XerR-overexpressing Xcc cells when they were vacuum infiltrated into cabbage plants. We also preliminarily characterized the host factor(s) involved in the above mentioned interactions between Xcc and the host plant, showing that a plant material(s) with molecular weight(s) less than 1 kDa abolished the binding of XerR to the xccR promoter, while the same material enhanced the binding of XccR to the luxXc box in the pip promoter. Taken together, our results implicate XerR in a new layer of the regulatory mechanism controlling the expression of the virulence-related xccR/pip locus and provide clues to the identification of plant signal molecules that interact with XerR and XccR to enhance the virulence of Xcc.

Identification and functional characterization of small non-coding RNAs in Xanthomonas oryzae pathovar oryzae.

BACKGROUND: Small non-coding RNAs (sRNAs) are regarded as important regulators in prokaryotes and play essential roles in diverse cellular processes. Xanthomonas oryzae pathovar oryzae (Xoo) is an important plant pathogenic bacterium which causes serious bacterial blight of rice. However, little is known about the number, genomic distribution and biological functions of sRNAs in Xoo. RESULTS: Here, we performed a systematic screen to identify sRNAs in the Xoo strain PXO99. A total of 850 putative non-coding RNA sequences originated from intergenic and gene antisense regions were identified by cloning, of which 63 were also identified as sRNA candidates by computational prediction, thus were considered as Xoo sRNA candidates. Northern blot hybridization confirmed the size and expression of 6 sRNA candidates and other 2 cloned small RNA sequences, which were then added to the sRNA candidate list. We further examined the expression profiles of the eight sRNAs in an hfq deletion mutant and found that two of them showed drastically decreased expression levels, and another exhibited an Hfq-dependent transcript processing pattern. Deletion mutants were obtained for seven of the Northern confirmed sRNAs, but none of them exhibited obvious phenotypes. Comparison of the proteomic differences between three of the ΔsRNA mutants and the wild-type strain by two-dimensional gel electrophoresis (2-DE) analysis showed that these sRNAs are involved in multiple physiological and biochemical processes. CONCLUSIONS: We experimentally verified eight sRNAs in a genome-wide screen and uncovered three Hfq-dependent sRNAs in Xoo. Proteomics analysis revealed Xoo sRNAs may take part in various metabolic processes. Taken together, this work represents the first comprehensive screen and functional analysis of sRNAs in rice pathogenic bacteria and facilitates future studies on sRNA-mediated regulatory networks in this important phytopathogen.

3-Acetonyl-3-hydroxyoxindole: a new inducer of systemic acquired resistance in plants.

Systemic acquired resistance (SAR) is an inducible defence mechanism which plays a central role in protecting plants from microbial pathogen attack. Guided by bioassays, a new chemical inducer of SAR was isolated from the extracts of Strobilanthes cusia and identified to be 3-acetonyl-3-hydroxyoxindole (AHO), a derivative of isatin. Tobacco plants treated with AHO exhibited enhanced resistance to tobacco mosaic virus (TMV) and to the fungal pathogen Erysiphe cichoracearum (powdery mildew), accompanied by increased levels of pathogenesis-related gene 1 (PR-1) expression, salicylic acid (SA) accumulation and phenylalanine ammonia-lyase activity. To study the mode of action of AHO, its ability to induce PR-1 expression and TMV resistance in nahG transgenic plants expressing salicylate hydroxylase, which prevents the accumulation of SA, was analysed. AHO treatment did not induce TMV resistance or PR-1 expression in nahG transgenic plants, suggesting that AHO acts upstream of SA in the SAR signalling pathway. In addition, using two-dimensional gel electrophoresis combined with mass spectrometry, five AHO-induced plant proteins were identified which were homologous to the effector proteins with which SA interacts. Our data suggest that AHO may represent a novel class of inducer that stimulates SA-mediated defence responses.