The Effect of Bacterial AHL on the Cyclic Adenosine Monophosphate Content in Plants According to High-Performance Liquid Chromatography.

Cyclic adenosine monophosphate (cAMP) is an important second messenger in cells, mediating various stimulation signals such as the growth and development of organisms and stress and participating in regulating various biological processes of cells. This article explores the quantitative determination of cAMP in plants using High-Performance Liquid Chromatography (HPLC) and applies this method to analyzing the changes in cAMP content during the process of plant response to the bacterial quorum sensing signal N-acyl homoserine lactone (AHL). Research has shown that the optimal detection conditions for HPLC are as follows: the chromatographic column is Venusil MP C18 (2), the mobile phase is methanol-water (0.1% trifluoroacetic acid) (v:v, 10:90), the detection wavelength is 259 nm, the column temperature is 35 °C, and the flow rate is 0.8 mL/min. The precision of the standard sample of this method is 98.21%, the precision of the sample is 98.87%, and the recovery rate is 101.067%. The optimal extraction conditions for cAMP in Arabidopsis are to use 15% methanol ultrasonic extraction for 10 min, followed by a 40 °C water bath for 4 h. Bacterial AHL signal processing can significantly stimulate an increase in cAMP levels in Arabidopsis leaves and roots. The establishment of HPLC detection methods for the cAMP content in plants is of great significance for in-depth research on the signal transduction mechanisms of plant-bacterial interactions.

Diffusible signal factor primes plant immunity against Xanthomonas campestris pv. campestris (Xcc) via JA signaling in Arabidopsis and Brassica oleracea.

Background: Many Gram-negative bacteria use quorum sensing (QS) signal molecules to monitor their local population density and to coordinate their collective behaviors. The diffusible signal factor (DSF) family represents an intriguing type of QS signal to mediate intraspecies and interspecies communication. Recently, accumulating evidence demonstrates the role of DSF in mediating inter-kingdom communication between DSF-producing bacteria and plants. However, the regulatory mechanism of DSF during the Xanthomonas-plant interactions remain unclear. Methods: Plants were pretreated with different concentration of DSF and subsequent inoculated with pathogen Xanthomonas campestris pv. campestris (Xcc). Pathogenicity, phynotypic analysis, transcriptome combined with metabolome analysis, genetic analysis and gene expression analysis were used to evaluate the priming effects of DSF on plant disease resistance. Results: We found that the low concentration of DSF could prime plant immunity against Xcc in both Brassica oleracea and Arabidopsis thaliana. Pretreatment with DSF and subsequent pathogen invasion triggered an augmented burst of ROS by DCFH-DA and DAB staining. CAT application could attenuate the level of ROS induced by DSF. The expression of RBOHD and RBOHF were up-regulated and the activities of antioxidases POD increased after DSF treatment followed by Xcc inoculation. Transcriptome combined with metabolome analysis showed that plant hormone jasmonic acid (JA) signaling involved in DSF-primed resistance to Xcc in Arabidopsis. The expression of JA synthesis genes (AOC2, AOS, LOX2, OPR3 and JAR1), transportor gene (JAT1), regulator genes (JAZ1 and MYC2) and responsive genes (VSP2, PDF1.2 and Thi2.1) were up-regulated significantly by DSF upon Xcc challenge. The primed effects were not observed in JA relevant mutant coi1-1 and jar1-1. Conclusion: These results indicated that DSF-primed resistance against Xcc was dependent on the JA pathway. Our findings advanced the understanding of QS signal-mediated communication and provide a new strategy for the control of black rot in Brassica oleracea.

Cellular messengers involved in the inhibition of the Arabidopsis primary root growth by bacterial quorum-sensing signal N-decanoyl-L-homoserine lactone.

BACKGROUND: N-acyl-homoserine lactones (AHLs) are used as quorum-sensing signals by Gram-negative bacteria, but they can also affect plant growth and disease resistance. N-decanoyl-L-homoserine lactone (C10-HSL) is an AHL that has been shown to inhibit primary root growth in Arabidopsis, but the mechanisms underlying its effects on root architecture are unclear. Here, we investigated the signaling components involved in C10-HSL-mediated inhibition of primary root growth in Arabidopsis, and their interplay, using pharmacological, physiological, and genetic approaches. RESULTS: Treatment with C10-HSL triggered a transient and immediate increase in the concentrations of cytosolic free Ca2+ and reactive oxygen species (ROS), increased the activity of mitogen-activated protein kinase 6 (MPK6), and induced nitric oxide (NO) production in Arabidopsis roots. Inhibitors of Ca2+ channels significantly alleviated the inhibitory effect of C10-HSL on primary root growth and reduced the amounts of ROS and NO generated in response to C10-HSL. Inhibition or scavenging of ROS and NO neutralized the inhibitory effect of C10-HSL on primary root growth. In terms of primary root growth, the respiratory burst oxidase homolog mutants and a NO synthase mutant were less sensitive to C10-HSL than wild type. Activation of MPKs, especially MPK6, was required for C10-HSL to inhibit primary root growth. The mpk6 mutant showed reduced sensitivity of primary root growth to C10-HSL, suggesting that MPK6 plays a key role in the inhibition of primary root growth by C10-HSL. CONCLUSION: Our results indicate that MPK6 acts downstream of ROS and upstream of NO in the response to C10-HSL. Our data also suggest that Ca2+, ROS, MPK6, and NO are all involved in the response to C10-HSL, and may participate in the cascade leading to C10-HSL-inhibited primary root growth in Arabidopsis.

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.

N-3-oxo-hexanoyl-homoserine lactone, a bacterial quorum sensing signal, enhances salt tolerance in Arabidopsis and wheat.

BACKGROUND: N-acyl-homoserine lactones (AHLs) are the quorum sensing (QS) signal molecules to coordinate the collective behavior in a population in Gram-negative bacteria. Recent evidences demonstrate their roles in plant growth and defense responses. RESULTS: In present study, we show that the treatment of plant roots with N-3-oxo-hexanoyl-homoserine lactone (3OC6-HSL), one molecule of AHLs family, resulted in enhanced salt tolerance in Arabidopsis and wheat. We found that the growth inhibition phenotype including root length, shoot length and fresh weight were significantly improved by 3OC6-HSL under salt stress condition. The physiological and biochemical analysis revealed that the contents of chlorophyll and proline were increased and the contents of MDA and Na+ and Na+/K+ ratios were decreased after 3OC6-HSL treatment in Arabidopsis and wheat under salt stress condition. Molecular analysis showed that 3OC6-HSL significantly upregulated the expression of salt-responsive genes including ABA-dependent osmotic stress responsive genes COR15a, RD22, ADH and P5CS1, ABA-independent gene ERD1, and ion-homeostasis regulation genes SOS1, SOS2 and SOS3 in Arabidopsis under salt stress condition. CONCLUSIONS: These results indicated that 3OC6-HSL enhanced plant salt tolerance and ABA-dependent and ABA-independent signal pathways and SOS signaling might be involved in the induction of salt resistance by 3OC6-HSL in plants. Our data provide a new insight into the plant-microbe inter-communication.

N-3-oxo-octanoyl-homoserine lactone-mediated priming of resistance to Pseudomonas syringae requires the salicylic acid signaling pathway in Arabidopsis thaliana.

BACKGROUD: Many Gram-negative bacteria use N-acyl-homoserine lactones (AHLs) to communicate each other and to coordinate their collective behaviors. Recently, accumulating evidence shows that host plants are able to sense and respond to bacterial AHLs. Once primed, plants are in an altered state that enables plant cells to more quickly and/or strongly respond to subsequent pathogen infection or abiotic stress. RESULTS: In this study, we report that pretreatment with N-3-oxo-octanoyl-homoserine lactone (3OC8-HSL) confers resistance against the pathogenic bacterium Pseudomonas syringae pv. tomato DC3000 (PstDC3000) in Arabidopsis. Pretreatment with 3OC8-HSL and subsequent pathogen invasion triggered an augmented burst of hydrogen peroxide, salicylic acid accumulation, and fortified expression of the pathogenesis-related genes PR1 and PR5. Upon PstDC3000 challenge, plants treated with 3OC8-HSL showed increased activities of defense-related enzymes including peroxidase, catalase, phenylalanine ammonialyase, and superoxide dismutase. In addition, the 3OC8-HSL-primed resistance to PstDC3000 in wild-type plants was impaired in plants expressing the bacterial NahG gene and in the npr1 mutant. Moreover, the expression levels of isochorismate synthases (ICS1), a critical salicylic acid biosynthesis enzyme, and two regulators of its expression, SARD1 and CBP60g, were potentiated by 3OC8-HSL pretreatment followed by pathogen inoculation. CONCLUSIONS: Our data indicate that 3OC8-HSL primes the Arabidopsis defense response upon hemibiotrophic bacterial infection and that 3OC8-HSL-primed resistance is dependent on the SA signaling pathway. These findings may help establish a novel strategy for the control of plant disease.

[Effects of different application methods of Bacillus subtilis agent on soil microbial diversity and growth of muskmelon].

Continuous planting of muskmelon and excessive application of chemical fertilizers have caused a series of problems, such as imbalance of the soil micro-ecological environment, serious soil-borne diseases and yield loss. Application of Bacillus subtilis agent is an important way to improve soil micro-ecological environment, prevent soil-borne diseases, and promote plant growth. In this study, B. subtilis was used as experimental agent to analyze the effects of different application methods on the soil microbial diversity and growth of muskmelon in greenhouse. The number of culturable microorganisms in soil was measured by dilution-plate method. The diversity of soil uncultivated microorganisms was determined by Illumina Miseq sequencing technology. The yield of muskmelon was measured by weighing method. The number of culturable bacteria in the root irrigation, hole application and dipping root application groups was higher than that of the control in different muskmelon growth stages, but there was no significant difference among the three different application methods. The number of soil fungi from B. subtilis agent treatment groups in flowering stage was significantly lower in comparison to the control group. However, B. subtilis agent treatment did not cause significant difference on soil fungi number at the fruiting and pulling stage. Diversity analysis of uncultured microorganisms showed that the Shannon index values of bacteria were higher and Simpson index values were lower respectively in the three B. subtilis treatment groups than that in the control. Moreover, the dipping root treatment produced the lowest Shannon index value and the highest Simpson index value of fungi. NMDS and cluster analysis showed that B. subtilis agents dipping root treatment significantly affected the bacterial and fungal flora, both of which were clustered into one independent branch. The application of B. subtilis agents, especially dipping root treatment, significantly decreased the abundance of Bacteroidetes, increased the abundance of Actinobacteria and Acidobacteria. The B. subtilis agent treatment didn't produce significant effect on the diversity of fungal flora except Chytridiomycota. The height, stem diameter and leaf area of muskmelon increased by applying B. subtilis agents, and dipping root treatment produced the most significant effect. As a new type of environmental protection fertilizer, B. subtilis agent can increase the number of soil culturable microorganisms, improve soil microbial diversity, and promote growth and yield. This study would provide a scientific basis for the rational application of B. subtilis.

Genomics and LC-MS Reveal Diverse Active Secondary Metabolites in Bacillus amyloliquefaciens WS-8.

Bacillus amyloliquefaciens is an important plant disease-preventing and growth-promoting microorganism. B. amyloliquefaciens WS-8 can stimulate plant growth and has strong antifungal properties. In this study, we sequenced the complete genome of B. amyloliquefaciens WS-8 by Pacific Biosciences RSII (PacBio) Single Molecule Real-Time (SMRT) sequencing. The genome consists of one chromosome (3,929,787 bp) and no additional plasmids. The main bacteriostatic substances were determined by genome, transcriptome, and mass spectrometry data. We thereby laid a theoretical foundation for the utilization of the strain. By genomic analysis, we identified 19 putative biosynthetic gene clusters for secondary metabolites, most of which are potentially involved in the biosynthesis of numerous bioactive metabolites, including difficidin, fengycin, and surfactin. Furthermore, a potential class II lanthipeptide biosynthetic gene cluster and genes that are involved in auxin biosynthesis were found. Through the analysis of transcriptome data, we found that the key bacteriostatic genes, as predicted in the genome, exhibited different levels of mRNA expression. Through metabolite isolation, purification, and exposure experiments, we found that a variety of metabolites of WS-8 exert an inhibitory effect on the necrotrophic fungus Botrytis cinerea, which causes gray mold; by mass spectrometry, we found that the main substances are mainly iturins and fengycins. Therefore, this strain has the potential to be utilized as an antifungal agent in agriculture.

Whole RNA-sequencing and gene expression analysis of Trichoderma harzianum Tr-92 under chlamydospore-producing condition.

BACKGROUND: Trichoderma is one of the most important biocontrol fungi, which could produce mycelia, conidiospores, and chlamydospores three types of propagules under different conditions. Chlamydospores are produced in harsh conditions in various fungi, and may be more resistant to adverse conditions. However, the knowledge associated with the mechanism of chlamydospore formation remained unclear in Trichoderma. OBJECTIVES: This study is aimed to explore the essential genes and regulatory pathways associated with chlamydospore formation in Trichoderma. METHODS: The culture condition, survival rate, and biocontrol effects of chlamydospores and conidiospores from Trichoderma.harzianum Tr-92 were determined. Furthermore, the whole transcriptome profiles of T. harzianum Tr-92 under chlamydospore-producing and chlamydospore-nonproducing conditions were performed. RESULTS: T. harzianum Tr-92 produced chlamydospores under particular conditions, and chlamydospore-based formulation of T. harzianum Tr-92 exhibited higher biocontrol ability against Botrytis cinerea in cucumber than conidoiospore-based formulation. In the transcriptome analysis, a total of 2,029 differentially expressed genes (DEGs) were identified in T. harzianum Tr-92 under chlamydospore-producing condition, compared to that under chlamydospore-nonproducing condition. GO classification indicated that the DEGs were significantly enriched in 284 terms among biological process, cellular components and molecular function categories. A total of 19 pathways were observed with DEGs by KEGG analysis. Furthermore, fifteen DEGs were verified by quantitative real-time PCR, and the expression profiles were consistent with the transcriptome data. CONCLUSION: The results would provide a basis on the molecular mechanisms underlying Trichoderma sporulation, which would assist the development and application of fungal biocontrol agents.

Involvement of jasmonic acid, ethylene and salicylic acid signaling pathways behind the systemic resistance induced by Trichoderma longibrachiatum H9 in cucumber.

BACKGROUND: Trichoderma spp. are effective biocontrol agents for many plant pathogens, thus the mechanism of Trichoderma-induced plant resistance is not fully understood. In this study, a novel Trichoderma strain was identified, which could promote plant growth and reduce the disease index of gray mold caused by Botrytis cinerea in cucumber. To assess the impact of Trichoderma inoculation on the plant response, a multi-omics approach was performed in the Trichoderma-inoculated cucumber plants through the analyses of the plant transcriptome, proteome, and phytohormone content. RESULTS: A novel Trichoderma strain was identified by morphological and molecular analysis, here named T. longibrachiatum H9. Inoculation of T. longibrachiatum H9 to cucumber roots promoted plant growth in terms of root length, plant height, and fresh weight. Root colonization of T. longibrachiatum H9 in the outer layer of epidermis significantly inhibited the foliar pathogen B. cinerea infection in cucumber. The plant transcriptome and proteome analyses indicated that a large number of differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were identified in cucumber plants 96 h post T. longibrachiatum H9 inoculation. Up-regulated DEGs and DEPs were mainly associated with defense/stress processes, secondary metabolism, and phytohormone synthesis and signaling, including jasmonic acid (JA), ethylene (ET) and salicylic acid (SA), in the T. longibrachiatum H9-inoculated cucumber plants in comparison to untreated plants. Moreover, the JA and SA contents significantly increased in cucumber plants with T. longibrachiatum H9 inoculation. CONCLUSIONS: Application of T. longibrachiatum H9 to the roots of cucumber plants effectively promoted plant growth and significantly reduced the disease index of gray mold caused by B. cinerea. The analyses of the plant transcriptome, proteome and phytohormone content demonstrated that T. longibrachiatum H9 mediated plant systemic resistance to B. cinerea challenge through the activation of signaling pathways associated with the phytohormones JA/ET and SA in cucumber.

Complete Genome Sequence of Bacillus subtilis J-5, a Potential Biocontrol Agent.

Bacillus subtilis J-5 was isolated from tomato rhizosphere soil and exhibited strong inhibitory activity against Botrytis cinerea To shed light on the molecular mechanism underlying the biological control on phytopathogens, the whole genome of this strain was sequenced. Genes encoding antimicrobial compounds and the regulatory systems were identified in the genome.

AtMYB44 Positively Regulates the Enhanced Elongation of Primary Roots Induced by N-3-Oxo-Hexanoyl-Homoserine Lactone in Arabidopsis thaliana.

N-acyl-homoserine lactones (AHL) are the quorum-sensing (QS) signal molecules used by many gram-negative bacteria to coordinate their collective behavior in a population. Recent evidence demonstrates their roles in plant root growth and defense responses. AtMYB44 is a multifaceted transcriptional factor that functions in many physiological processes in plants but whether AtMYB44 modulates the plant response to AHL with aspects of primary root elongation remains unknown. Here, we show that the expression of AtMYB44 was upregulated upon treatment with N-3-oxo-hexanoyl-homoserine lactone (3OC6-HSL). The stimulatory effect of 3OC6-HSL on primary root elongation was abolished in the AtMYB44 functional-deficiency mutant atmby44. In contrast, an enhanced promoting-impact of 3OC6-HSL on primary root growth was observed in AtMYB44-overexpressing plant MYB44OTA. Cellular analysis indicated that the prolonged primary root elicited by 3OC6-HSL is the consequence of increased cell division in the meristem zone and enhanced cell elongation in the elongation zone, and AtMYB44 may act as a positive regulator in this process. Furthermore, we demonstrated that AtMYB44 might participate in the 3OC6-HSL-mediated primary root growth via regulating the expression of cytokinin- and auxin-related genes. The data establish a genetic connection between the regulatory role of AtMYB44 in phytohormones-related gene expression and plant response to the bacterial QS signal.

Involvement of calmodulin in regulation of primary root elongation by N-3-oxo-hexanoyl homoserine lactone in Arabidopsis thaliana.

Many bacteria use signal molecules of low molecular weight to monitor their local population density and to coordinate their collective behavior in a process called "quorum sensing" (QS). N-acyl-homoserine lactones (AHLs) are the primary QS signals among Gram-negative bacteria. AHL-mediated QS plays an essential role in diverse bacterial physiological processes. Recent evidence shows that plants are able to sense bacterial AHLs and respond to them appropriately. However, little is known about the mechanism by which plants perceive and transduce the bacterial AHLs within cells. In this study, we found that the stimulatory effect of N-3-oxo-hexanoyl homoserine lactone (3OC6-HSL) on primary root elongation of Arabidopsis was abolished by the calmodulin (CaM) antagonists N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7) and trifluoperazine (TFP). Western-blot and ELISA analysis revealed that the concentration of CaM protein in Arabidopsis roots increased after treatment with 1 µM 3OC6-HSL. Results from quantitative RT-PCR demonstrated that the transcription of all nine CaM genes in Arabidopsis genome was up-regulated in the plants treated with 3OC6-HSL. The loss-of-function mutants of each AtCaM gene (AtCaM1-9) were insensitive to 3OC6-HSL-stimulation of primary root elongation. On the other hand, the genetic evidence showed that CaM may not participates the inhibition of primary root length caused by application of long-chained AHLs such as C10-HSL and C12-HSL. Nevertheless, our results suggest that CaM is involved in the bacterial 3OC6-HSL signaling in plant cells. These data offer new insight into the mechanism of plant response to bacterial QS signals.

A proteomic analysis of Arabidopsis thaliana seedling responses to 3-oxo-octanoyl-homoserine lactone, a bacterial quorum-sensing signal.

N-acyl-homoserine lactones (AHLs) are a class of bacterial quorum-sensing (QS) signals that are commonly used by Gram-negative bacteria for cell-to-cell communication. Recently, it has become evident that AHLs can regulate plant root growth and trigger plant defense responses; however, little is known about the plant response mechanisms to bacterial QS signals. In this study, we used a proteomic approach to investigate the responses of Arabidopsis thaliana seedlings to N-3-oxo-octanoyl-homoserine lactone (3OC8-HSL), a bacterial QS signal. The results revealed that the abundance of 53 protein spots was significantly altered; two thirds of these proteins were found to be up-regulated after 3OC8-HSL treatment. Thirty-four proteins were identified using MALDI-TOF-MS. These 3OC8-HSL-responsive proteins, in addition to one protein of unknown function, are implicated in a variety of physiological processes, including metabolism of carbohydrate and energy, protein biosynthesis and quality control systems, defense response and signal transduction and cytoskeleton remodeling. Our bioinformatic analysis indicated that the chloroplasts are the intracellular organelles most influenced by the exposure to 3OC8-HSL. Our data indicate that plants have an extensive range of functional responses to bacterial AHLs that may play important roles in the interaction between plants and bacteria.

The GCR1 and GPA1 participate in promotion of Arabidopsis primary root elongation induced by N-acyl-homoserine lactones, the bacterial quorum-sensing signals.

Many gram-negative bacteria use N-acyl-homoserine lactones (AHL) as quorum-sensing signals to coordinate their collective behaviors. Accumulating evidence indicates that plants can respond to AHL. However, little is known about the molecular mechanism of plants reacting to these bacterial signals. In this study, we show that the treatment of Arabidopsis roots with N-3-oxo-hexanoyl-homoserine lactone (3OC6-HSL) and N-3-oxo-octanoyl-homoserine lactone (3OC8-HSL) resulted in significant root elongation. The genetic analysis revealed that the T-DNA insertional mutants of gcr1, encoding a G-protein-coupled receptor GCR1, were insensitive to 3OC6-HSL or 3OC8-HSL in assays of root growth. The loss-of-function mutants of the sole canonical Gα subunit GPA1 showed no response to AHL promotion of root elongation whereas Gα gain-of-function plants overexpressing either the wild type or a constitutively active version of Arabidopsis Gα exhibited the exaggerated effect on root elongation caused by AHL. Furthermore, the expression of GCR1 and GPA1 were significantly upregulated after plants were contacted with both AHL. Taken together, our results suggest that GCR1 and GPA1 are involved in AHL-mediated elongation of Arabidopsis roots. This provides insight into the mechanism of plant responses to bacterial quorum-sensing signals.

Two G-protein-coupled-receptor candidates, Cand2 and Cand7, are involved in Arabidopsis root growth mediated by the bacterial quorum-sensing signals N-acyl-homoserine lactones.

Many Gram-negative bacteria use N-acyl-homoserine lactones (AHLs) as quorum sensing (QS) signaling molecules to coordinate their group behavior. Recently, it was shown that plants can perceive and respond to these bacterial AHLs. However, little is known about the molecular mechanism underlying the response of plants to bacterial QS signals. In this study, we show that the promotion of root elongation in wild type Arabidopsis thaliana induced by the AHLs N-3-oxo-hexanoyl-homoserine lactone (3OC6-HSL) or N-3-oxo-octanoyl-homoserine lactone (3OC8-HSL) was completely abolished in plants with loss-of-function mutations in two candidate G-protein Coupled Receptors (GPCRs), Cand2 and Cand7. Furthermore, real-time PCR analysis revealed that the expression levels of Cand2 and Cand7 were elevated in plants treated with 3OC6-HSL or 3OC8-HSL. These results suggest that Cand2 and Cand7 are involved in the regulation of root growth by bacterial AHLs and that GPCRs play a role in mediating interactions between plants and microbes.

N-butyryl-homoserine lactone, a bacterial quorum-sensing signaling molecule, induces intracellular calcium elevation in Arabidopsis root cells.

N-acyl-L-homoserine lactones (AHLs) are quorum sensing (QS) signal molecules that are commonly used in gram-negative bacteria. Recently, it has become evident that AHLs can influence the behavior of plant cells. However, little is known about the mechanism of the plants' response to these bacterial signals. Calcium ions (Ca(2+)), ubiquitous intracellular second messengers, play an essential role in numerous signal transduction pathways in plants. In this study, the cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) was measured by a luminometric method in the excised root cells of Arabidopsis plants that were treated with N-butyryl-homoserine lactone (C4-HSL). There was a transient and immediate increase in [Ca(2+)](cyt) levels, and the highest level (0.4 µM), approximately 2-fold higher than the basal level, was observed at the 6th second after the addition of 10 µM C4-HSL. Pretreatments with La(3+), verapamil or ethylene glycol tetraacetic acid (EGTA) inhibited the increase in [Ca(2+)](cyt) caused by C4-HSL, whereas it remained unaffected by pretreatment with Li(+), indicating that the Ca(2+) contributing to the increase in [Ca(2+)](cyt) was mobilized from the extracellular medium via the plasma membrane Ca(2+) channels but not from the intracellular Ca(2+) stores. Furthermore, electrophysiological approaches showed that the transmembrane Ca(2+) current was significantly increased with the addition of C4-HSL. Taken together, our observations suggest that C4-HSL may act as an elicitor from bacteria to plants and that Ca(2+) signaling participates in the ability of plant cells to sense the bacterial QS signals.

[Involvement of quorum-sensing in biosynthesis of polyhydroxyalkanoates in Pseudomonas aeruginosa].

UNLABELLED: Quorum-sensing (QS) is a regulatory mechanism with which bacteria regulate the gene expression according to their population density. Pseudomonas aeruginosa regulates the expression of multiple genes via a hierarchical quorum-sensing cascade through LasR and RhlR and their cognate signal molecules N-(3-oxododecanoyl)-L-homoserine lactone (30-C12-HSL) and N-(butanoyl)-L-homoserine lactone (C4-HSL). OBJECTIVE: It aims to explore the regulation of QS on biosynthesis of polyhydroxyalkanoates (PHA) in P. aeruginosa. METHODS: Wild-type P. aeruginosa PA01 and its QS mutants were used to investigate the effects of quorum-sensing on biosynthesis of PHA by GC and real-time PCR at physiological and molecular level. RESULTS: After treated with QS signal molecule synthesis inhibitor azithromycin, the accumulation of PHA significantly decreased in P. aeruginosa PA01 and its QS mutant strains. The content of PHA in C4-HSL synthase gene rhlI mutant strain PA0210 had no significant difference compared with that of the wild type. However, the PHA contents were significantly affected in 30C12-HSL synthase gene lasl mutant strain PA055, 30C12-HSL transcriptional regulator gene lasR mutant strain PA056 and lasI/lasR double mutant strain PA057. PHA synthase gene phaC1 expression exhibited a significant reduction in lasI mutant and lasR mutant strains. 30C12-HSL signal molecules complementary experiment shows that the expression of phaC1 can be recovered to the level of the wild type, but the synthesis of PHA is only partially restored in lasI mutant strain. CONCLUSION: The results implicates that lasI/lasR system might be involved in the regulation of intracellular PHA biosynthesis in P. aeruginosa PA01.

Quercetin-induced H(2)O(2) mediates the pathogen resistance against Pseudomonas syringae pv. Tomato DC3000 in Arabidopsis thaliana.

Quercetin is a potent antioxidant and has been extensively used as a therapy intervention to prevent age-associated diseases. However, emerging studies showed it can also act as a prooxidant and induce H(2)O(2) under certain conditions. In the current study, our results showed that quercetin contributed to the pathogen resistance in Arabidopsis thaliana (Arabidopsis) in response to the infection of virulent strain Pseudomonas syringae pv. Tomato DC3000 (Pst). Various defense responses, such as H(2)O(2) burst, callose deposition, cell death, PR1 (pathogenesis-related 1) and PAL1 (Phe ammonia-lyase 1) gene expression, have been investigated in quercetin-pretreated Pst-inoculated Arabidopsis Col-0 and there was a strong defensive response in quercetin-pretreated Arabidopsis against virulent Pst. However, with the presence of catalase, the protective effects of quercetin on pathogen resistance to virulent Pst disappeared in Arabidopsis, suggesting that H(2)O(2) may play a key role in plant defense responses. In addition, we confirmed that quercetin did not show any beneficial effect on pathogen-free leaves in Arabidopsis, indicating that pathogen challenge is also required to induce the defense responses in quercetin-pretreated Arabidopsis. Furthermore, strong defense responses have been observed in quercetin-pretreated Arabidopsis mutant jar1, ein2, and abi1-2 under Pst challenge, whereas no protective effect has been observed in quercetin-pretreated Arabidopsis mutant NahG and npr1. These findings indicate that quercetin induces the resistance to Pst in Arabidopsis via H(2)O(2) burst and involvement of SA and NPR1.

[Isolation and identification of Rhodosporidium toruloides R1 degrading N-acyl homoserine lactone].

In the present study, a AHL-utilizing strain RI was isolated and identified as the genus Rhodosporidium toruloides R1 by physi-biochemical approaches and 18S rDNA sequence analysis, and this strain was designated as R. toruloides R1. Results showed that R. toruloides R1 exhibited the ability to utilize and degrade the all N-acyl homoserine lactones tested in this study. Coculture of R. toruloides R1 with Erwinia carotovora subsp. Carotovora effectively inhibit the soft rot disease of patato caused by E. carotovora. To the best of our knowledge, this is the first report on AHL-degradation of yeast cells.