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The MIR396 family, composed of ath-miR396a and ath-miR396b in Arabidopsis, is conserved among plant species and is known to target the Growth-Regulating Factor ( GRF) gene family. ath-miR396 overexpressors or grf mutants are characterised by small and narrow leaves and show embryogenic defects such as cotyledon fusion. Heterologous expression of ath-miR396a has been reported in tobacco and resulted in reduction of the expression of three Nt GRF genes. In this study, the precursor of the Populus trichocarpa ptc-miR396c, with a mature sequence identical to ath-miR396b, was expressed under control of the Ca MV35S promoter in tobacco. Typical phenotypes of GRF down-regulation were observed, including cotyledon fusion and lack of shoot apical meristem ( SAM). At later stage of growth, transgenic plants had delayed development and altered specification of organ type during flower development. The third and fourth whorls of floral organs were modified into stigmatoid anthers and fasciated carpels, respectively. Several Nt GRF genes containing a miR396 binding site were found to be down-regulated, and the cleavage of their corresponding m RNA at the miR396 binding site was confirmed for two of them using RACE- PCR analysis. The data obtained agree with the functional conservation of the miR396 family in plants and suggest a role for the miR396/ GRF network in determination of floral organ specification. [ABSTRACT FROM AUTHOR]

Psidium guajava is one of the most common edible medicinal plants frequently used in Malagasy traditional medicine to treat gastrointestinal infections. In order to evaluate their probable antibacterial activities, three organic extracts (successive extractions by hexane, dichloromethane, and ethanol) of ripe guava fruits were assessed for their bactericidal and anti-virulence properties against P. aeruginosa PAO1. Although these three extracts have shown no direct antibacterial activity (MIC of 1000 µg/mL) and, at the non-bactericidal concentration of 100 µg/mL, no impact on the production of major P. aeruginosa PAO1 virulence factors (pyocyanin and rhamnolipids), the hexane and dichloromethane extracts showed significant anti-biofilm properties and the dichloromethane extract disrupted the P. aeruginosa PAO1 swarming motility. Bioguided fractionation of the dichloromethane extract led to the isolation and identification of lycopene and β-sitosterol-β-D-glucoside as major anti-biofilm compounds. Interestingly, both compounds disrupt P. aeruginosa PAO1 biofilm formation and maintenance with IC 50 of 1383 µM and 131 µM, respectively. More interestingly, both compounds displayed a synergistic effect with tobramycin with a two-fold increase in its effectiveness in killing biofilm-encapsulated P. aeruginosa PAO1. The present study validates the traditional uses of this edible medicinal plant, indicating the therapeutic effectiveness of guava fruits plausibly through the presence of these tri- and tetraterpenoids, which deserve to be tested against pathogens generally implicated in diarrhea.

Introduction: Plant A/T-rich protein and zinc-binding protein (PLATZ) are plant-specific transcription factors playing a role in plant development and stress response. To assess the role of PLATZs in vascular system development and wood formation in poplar, a functional study for PtaPLATZ18 , whose expression was associated with the xylem, was carried out., Methods: Poplar dominant repressor lines for PtaPLATZ18 were produced by overexpressing a PtaPLATZ18-SRDX fusion. The phenotype of three independent transgenic lines was evaluated at morphological, biochemical, and molecular levels and compared to the wild type., Results: The PtaPLATZ18-SRDX lines showed increased plant height resulting from higher internode length. Besides, a higher secondary xylem thickness was also evidenced in these dominant repression lines as compared to the wild type suggesting an activation of cambial activity. A higher amount of lignin was evidenced within wood tissue as compared to the wild type, indicating an alteration in cell wall composition within xylem cell types. This latter phenotype was linked to an increased expression of genes involved in lignin biosynthesis and polymerization., Discussion: The phenotype observed in the PtaPLATZ18-SRDX lines argues that this transcription factor targets key regulators of plant growth and vascular tissues development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Guérin, Behr, Sait, Mol, El Jaziri and Baucher.)

In some specific vascular plant tissues, lignin can impregnate the entire cell wall to make it more rigid and hydrophobic. Different techniques have been developed in the past years to make possible the quantification of this polyphenolic polymer at the organ or tissue level, but difficulties of access to the cellular level remain. Here we describe an approach based on ratiometric emission measurements using safranin-O and the development of a macro adapted for the FIJI software, which makes it possible to quantify lignin in three different layers of the cell wall on images captured on a fluorescent confocal microscope., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Recently, the xanthophyll carotenoid lutein has been qualified as a potential quorum sensing (QS) and biofilm inhibitor against Pseudomonas aeruginosa. To address the potential of this xanthophyll compound as a relevant antivirulence agent, we investigated in depth its impact on the invasion capabilities and aggressiveness of P. aeruginosa PAO1, which rely on the bacterial ability to build and maintain protective barriers, use different types of motilities and release myriad virulence factors, leading to host cell and tissue damages. Our data, obtained on the PAO1 strain, indicate that all- trans lutein (Lut; 22 µM) disrupts biofilm formation and disorganizes established biofilm structure without affecting bacterial viability, while improving the bactericidal activity of tobramycin against biofilm-encapsulated PAO1 cells. Furthermore, this xanthophyll affects PAO1 twitching and swarming motilities while reducing the production of the extracellular virulence factors pyocyanin, elastase and rhamnolipids as well as the expression of the QS-regulated lasB and rhlA genes without inhibiting the QS-independent aceA gene. Interestingly, the expression of the QS regulators rhlR / I and lasR / I is significantly reduced as well as that of the global virulence factor regulator vfr , which is suggested to be a major target of Lut. Finally, an oxidative metabolite of Lut, 3'-dehydrolutein, induces a similar inhibition phenotype. Taken together, lutein-type compounds represent potential agents to control the invasive ability and antibiotic resistance of P. aeruginosa .

Reactive species (RS) causing oxidative stress are unavoidable by-products of various plant metabolic processes, such as photosynthesis, respiration or photorespiration. In leaves, flavonoids scavenge RS produced during photosynthesis and protect plant cells against deleterious oxidative damages. Their biosynthesis and accumulation are therefore under tight regulation at the cellular level. Glycosylation has emerged as an essential biochemical reaction in the homeostasis of various specialized metabolites such as flavonoids. This article provides a functional characterization of the Populus tremula x P. alba (poplar) UGT72A2 coding for a UDP-glycosyltransferase that is localized in the chloroplasts. Compared with the wild type, transgenic poplar lines with decreased expression of UGT72A2 are characterized by reduced growth and oxidative damages in leaves, as evidenced by necrosis, higher content of glutathione and lipid peroxidation products as well as diminished soluble peroxidase activity and NADPH to NADP+ ratio under standard growing conditions. They furthermore display lower pools of phenolics, anthocyanins and total flavonoids but higher proanthocyanidins content. Promoter analysis revealed the presence of cis-elements involved in photomorphogenesis, chloroplast biogenesis and flavonoid biosynthesis. The UGT72A2 is regulated by the poplar MYB119, a transcription factor known to regulate the flavonoid biosynthesis pathway. Phylogenetic analysis and molecular docking suggest that UGT72A2 could glycosylate flavonoids; however, the actual substrate(s) was not consistently evidenced with either in vitro assays nor analyses of glycosylated products in leaves of transgenic poplar overexpressing or downregulated for UGT72A2. This article provides elements highlighting the importance of flavonoid glycosylation regarding protection against oxidative stress in poplar leaves and raises new questions about the link between this biochemical reaction and regulation of the redox homeostasis system., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Plants have developed the capacity to produce a diversified range of specialized metabolites. The glycosylation of those metabolites potentially decreases their toxicity while increasing their stability and their solubility, modifying their transport and their storage. The UGT, forming the largest glycosyltransferase superfamily in plants, combine enzymes that glycosylate mainly hormones and phenylpropanoids by using UDP-sugar as a sugar donor. Particularly, members of the UGT72 family have been shown to glycosylate the monolignols and the flavonoids, thereby being involved in their homeostasis. First, we explore primitive UGTs in algae and liverworts that are related to the angiosperm UGT72 family and their role in flavonoid homeostasis. Second, we describe the role of several UGT72s glycosylating monolignols, some of which have been associated with lignification. In addition, the role of other UGT72 members that glycosylate flavonoids and are involved in the development and/or stress response is depicted. Finally, the importance to explore the subcellular localization of UGTs to study their roles in planta is discussed.

Oxidative stress is a cellular threat which puts at risk the productivity of most of crops valorized by humankind in terms of food, feed, biomaterial, or bioenergy. It is therefore of crucial importance to understand the mechanisms by which plants mitigate the deleterious effects of oxidizing agents. Glycosylation of antioxidant molecules and phytohormones modifies their chemical properties as well as their cellular and histological repartition. This review emphasizes the mechanisms and the outcomes of this conjugation reaction on plant ability to face growing conditions favoring oxidative stress, in mirror with the activity of deglycosylating enzymes. Pioneer evidence bridging flavonoid, glycosylation, and redox homeostasis paved the way for numerous functional analyses of UDP-glycosyltransferases (UGTs), such as the identification of their substrates and their role to circumvent oxidative stress resulting from various environmental challenges. (De)glycosylation appears as a simple chemical reaction regulating the biosynthesis and/or the activity of a myriad of specialized metabolites partaking in response to pathogen and abiotic stresses. This outcome underlies the possibility to valorize UGTs potential to upgrade plant adaptation and fitness in a rising context of sub-optimal growing conditions subsequent to climate change., (Copyright © 2020 Behr, Neutelings, El Jaziri and Baucher.)

This study investigates the impact of the alteration of the monolignol biosynthesis pathway on the establishment of the in vitro interaction of poplar roots either with a mutualistic ectomycorrhizal fungus or with a pathogenic root-knot nematode. Overall, the five studied transgenic lines downregulated for caffeoyl-CoA O-methyltransferase (CCoAOMT), caffeic acid O-methyltransferase (COMT), cinnamoyl-CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD) or both COMT and CAD displayed a lower mycorrhizal colonisation percentage, indicating a lower ability for establishing mutualistic interaction than the wild-type. The susceptibility to root-knot nematode infection was variable in the five lines, and the CAD-deficient line was found to be less susceptible than the wild-type. We discuss these phenotypic differences in the light of the large shifts in the metabolic profile and gene expression pattern occurring between roots of the CAD-deficient line and wild-type. A role of genes related to trehalose metabolism, phytohormones, and cell wall construction in the different mycorrhizal symbiosis efficiency and nematode sensitivity between these two lines is suggested. Overall, these results show that the alteration of plant metabolism caused by the repression of a single gene within phenylpropanoid pathway results in significant alterations, at the root level, in the response towards mutualistic and pathogenic associates. These changes may constrain plant fitness and biomass production, which are of economic importance for perennial industrial crops such as poplar.

Lignin is present in plant secondary cell walls and is among the most abundant biological polymers on Earth. In this work we investigated the potential role of the UGT72E gene family in regulating lignification in Arabidopsis . Chemical determination of floral stem lignin contents in ugt72e1 , ugt72e2, and ugt72e3 mutants revealed no significant differences compared to WT plants. In contrast, the use of a novel safranin O ratiometric imaging technique indicated a significant increase in the cell wall lignin content of both interfascicular fibers and xylem from young regions of ugt72e3 mutant floral stems. These results were globally confirmed in interfascicular fibers by Raman microspectroscopy. Subsequent investigation using a bioorthogonal triple labelling strategy suggested that the augmentation in lignification was associated with an increased capacity of mutant cell walls to incorporate H-, G-, and S-monolignol reporters. Expression analysis showed that this increase was associated with an up-regulation of LAC17 and PRX71 , which play a key role in lignin polymerization. Altogether, these results suggest that UGT72E3 can influence the kinetics of lignin deposition by regulating monolignol flow to the cell wall as well as the potential of this compartment to incorporate monomers into the growing lignin polymer.

Monolignols are the building blocks for lignin polymerization in the apoplastic domain. Monolignol biosynthesis, transport, storage, glycosylation, and deglycosylation are the main biological processes partaking in their homeostasis. In Arabidopsis thaliana , members of the uridine diphosphate-dependent glucosyltransferases UGT72E and UGT72B subfamilies have been demonstrated to glycosylate monolignols. Here, the poplar UGT72 family, which is clustered into four groups, was characterized: Group 1 UGT72AZ1 and UGT72AZ2, homologs of Arabidopsis UGT72E1-3, as well as group 4 UGT72B37 and UGT72B39, homologs of Arabidopsis UGT72B1-3, glycosylate monolignols. In addition, promoter-GUS analyses indicated that poplar UGT72 members are expressed within vascular tissues. At the subcellular level, poplar UGT72s belonging to group 1 and group 4 were found to be associated with the nucleus and the endoplasmic reticulum. However, UGT72A2, belonging to group 2, was localized in bodies associated with chloroplasts, as well as possibly in chloroplasts. These results show a partial conservation of substrate recognition between Arabidopsis and poplar homologs, as well as divergent functions between different groups of the UGT72 family, for which the substrates remain unknown.

One of the main characteristics of plant cells is the presence of the cell wall located outside the plasma membrane. In particular cells, this wall can be reinforced by lignin, a polyphenolic polymer that plays a central role for vascular plants, conferring hydrophobicity to conducting tissues and mechanical support for upright growth. Lignin has been studied extensively by a range of different techniques, including anatomical and morphological analyses using dyes to characterize the polymer localization in situ. With the constant improvement of imaging techniques, it is now possible to revisit old qualitative techniques and adapt them to obtain efficient, highly resolutive, quantitative, fast and safe methodologies. In this study, we revisit and exploit the potential of fluorescent microscopy coupled to safranin-O staining to develop a quantitative approach for lignin content determination. The developed approach is based on ratiometric emission measurements and the development of an imagej macro. To demonstrate the potential of our methodology compared with other commonly used lignin reagents, we demonstrated the use of safranin-O staining to evaluate and compare lignin contents in previously characterized Arabidopsis thaliana lignin biosynthesis mutants. In addition, the analysis of lignin content and spatial distribution in the Arabidopsis laccase mutant also provided new biological insights into the effects of laccase gene downregulation in different cell types. Our safranin-O-based methodology, also validated for Linum usitatissimum (flax), Zea mays (maize) and Populus tremula x alba (poplar), significantly improves and speeds up anatomical and developmental investigations of lignin, which we hope will contribute to new discoveries in many areas of cell wall plant research., (© 2020 The Authors The Plant Journal © 2020 John Wiley & Sons Ltd.)

One of the most striking features occurring in the root-knot nematode Meloidogyne incognita induced galls is the reorganization of the vascular tissues. During the interaction of the model tree species Populus and M. incognita , a pronounced xylem proliferation was previously described in mature galls. To better characterise changes in expression of genes possibly involved in the induction and the formation of the de novo developed vascular tissues occurring in poplar galls, a comparative transcript profiling of 21-day-old galls versus uninfected root of poplar was performed. Genes coding for transcription factors associated with procambium maintenance and vascular differentiation were shown to be differentially regulated, together with genes partaking in phytohormones biosynthesis and signalling. Specific signatures of transcripts associated to primary cell wall biosynthesis and remodelling, as well as secondary cell wall formation (cellulose, xylan and lignin) were revealed in the galls. Ultimately, we show that molecules derived from the monolignol and salicylic acid pathways and related to secondary cell wall deposition accumulate in mature galls.

Although lignin is essential to ensure the correct growth and development of land plants, it may be an obstacle to the production of lignocellulosics-based biofuels, and reduces the nutritional quality of crops used for human consumption or livestock feed. The need to tailor the lignocellulosic biomass for more efficient biofuel production or for improved plant digestibility has fostered considerable advances in our understanding of the lignin biosynthetic pathway and its regulation. Most of the described regulators are transcriptional activators of lignin biosynthesis, but considerably less attention has been devoted to the repressors of this pathway. We provide a comprehensive overview of the molecular factors that negatively impact on the lignification process at both the transcriptional and post-transcriptional levels., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Plant root-knot nematode (RKN) interaction studies are performed on several host plant models. Though RKN interact with trees, no perennial woody model has been explored so far. Here, we show that poplar (Populus tremula × P. alba) grown in vitro is susceptible to Meloidogyne incognita, allowing this nematode to penetrate, to induce feeding sites, and to successfully complete its life cycle. Quantitative reverse transcription-polymerase chain reaction analysis was performed to study changes in poplar gene expression in galls compared with noninfected roots. Three genes (expansin A, histone 3.1, and asparagine synthase), selected as gall development marker genes, followed, during poplar-nematode interaction, a similar expression pattern to what was described for other plant hosts. Downregulation of four genes implicated in the monolignol biosynthesis pathway was evidenced in galls, suggesting a shift in the phenolic profile within galls developed on poplar roots. Raman microspectroscopy demonstrated that cell walls of giant cells were not lignified but mainly composed of pectin and cellulose. The data presented here suggest that RKN exercise conserved strategies to reproduce and to invade perennial plant species and that poplar is a suitable model host to study specific traits of tree-nematode interactions.

Background/aims: The Escherichia coli MazF is an endoribonuclease that cleaves mRNA at ACA sequences, thereby triggering inhibition of protein synthesis. The aim of this study is to evaluate the efficiency of the mazEF toxin-antitoxin system in plants to develop biotechnological tools for targeted cell ablation., Methods: A double transformation strategy, combining expression of the mazE antitoxin gene under the control of the CaMV 35S promoter, reported to drive expression in all plant cells except within the tapetum, together with the expression of the mazF gene under the control of the TA29 tapetum-specific promoter in transgenic tobacco, was applied., Results: No transgenic TA29-mazF line could be regenerated, suggesting that the TA29 promoter is not strictly tapetum specific and that MazF is toxic for plant cells. The regenerated 35S-mazE/TA29-mazF double-transformed lines gave a unique phenotype where the tapetal cell layer was necrosed resulting in the absence of pollen., Conclusion: These results show that the E. colimazEF system can be used to induce death of specific plant cell types and can provide a new tool to plant cell ablation., (© 2016 S. Karger AG, Basel.)

REALLY INTERESTING NEW GENE (RING) proteins play important roles in the regulation of many processes by recognizing target proteins for ubiquitination. Previously, we have shown that the expression of PtaRHE1, encoding a Populus tremula × Populus alba RING-H2 protein with E3 ubiquitin ligase activity, is associated with tissues undergoing secondary growth. To further elucidate the role of PtaRHE1 in vascular tissues, we have undertaken a reverse genetic analysis in poplar. Within stem secondary vascular tissues, PtaRHE1 and its corresponding protein are expressed predominantly in the phloem. The downregulation of PtaRHE1 in poplar by artificial miRNA triggers alterations in phloem fibre patterning, characterized by an increased portion of secondary phloem fibres that have a reduced cell wall thickness and a change in lignin composition, with lower levels of syringyl units as compared with wild-type plants. Following an RNA-seq analysis, a biological network involving hormone stress signalling, as well as developmental processes, could be delineated. Several candidate genes possibly associated with the altered phloem fibre phenotype observed in amiRPtaRHE1 poplar were identified. Altogether, our data suggest a regulatory role for PtaRHE1 in secondary phloem fibre development., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

Members of Gram-positive Actinobacteria cause economically important diseases to plants. Within the Rhodococcus genus, some members can cause growth deformities and persist as pathogens on a wide range of host plants. The current model predicts that phytopathogenic isolates require a cluster of three loci present on a linear plasmid, with the fas operon central to virulence. The Fas proteins synthesize, modify, and activate a mixture of growth regulating cytokinins, which cause a hormonal imbalance in plants, resulting in abnormal growth. We sequenced and compared the genomes of 20 isolates of Rhodococcus to gain insights into the mechanisms and evolution of virulence in these bacteria. Horizontal gene transfer was identified as critical but limited in the scale of virulence evolution, as few loci are conserved and exclusive to phytopathogenic isolates. Although the fas operon is present in most phytopathogenic isolates, it is absent from phytopathogenic isolate A21d2. Instead, this isolate has a horizontally acquired gene chimera that encodes a novel fusion protein with isopentyltransferase and phosphoribohydrolase domains, predicted to be capable of catalyzing and activating cytokinins, respectively. Cytokinin profiling of the archetypal D188 isolate revealed only one activate cytokinin type that was specifically synthesized in a fas-dependent manner. These results suggest that only the isopentenyladenine cytokinin type is synthesized and necessary for Rhodococcus phytopathogenicity, which is not consistent with the extant model stating that a mixture of cytokinins is necessary for Rhodococcus to cause leafy gall symptoms. In all, data indicate that only four horizontally acquired functions are sufficient to confer the trait of phytopathogenicity to members of the genetically diverse clade of Rhodococcus.

RING (REALLY INTERESTING NEW GENE) proteins with E3 ligase activity are largely represented in plants. They have been shown to play important roles in the regulation of many biological processes by recognizing target proteins for ubiquitination. PtaRHE1, encoding a poplar RING-H2 domain-containing protein with E3 ligase activity has been previously shown to be expressed during the establishment of secondary vascular system in poplar. In the present report, we demonstrate that the expression of PtaRHE1 and the accumulation of its corresponding protein are modulated by the relative atmospheric and soil humidity and by abscisic acid. Overall, the integrated data are discussed within a working model highlighting a plausible function of PtaRHE1 in the signaling and/or in the regulation of water status in poplar.

The multifunctionality of plant annexins and their importance for coordinating development and responses to biotic and abiotic environment have been largely reviewed. We recently described a tobacco annexin, named Ntann12, which is mainly localized in the nucleus of root cells when the plant is grown under light conditions. We also found that auxin and polar auxin transport are essential for Ntann12 accumulation in root cells. Under dark condition, Ntann12 is no longer detected in the root system. In the present addendum, light, regulating auxin signaling, is evidenced as an essential determinant for the synchronization of growth and development between the shoot and the root during light/dark cycle. A speculative model for Ntann12 is described and discussed with regards to relevant literature data.

Preliminary screening of the Malagasy plant Combretum albiflorum for compounds attenuating the production of quorum sensing (QS)-controlled virulence factors in bacteria led to the identification of active fractions containing flavonoids. In the present study, several flavonoids belonging to the flavone, flavanone, flavonol and chalcone structural groups were screened for their capacity to reduce the production of QS-controlled factors in the opportunistic pathogen Pseudomonas aeruginosa (strain PAO1). Flavanones (i.e. naringenin, eriodictyol and taxifolin) significantly reduced the production of pyocyanin and elastase in P. aeruginosa without affecting bacterial growth. Consistently, naringenin and taxifolin reduced the expression of several QS-controlled genes (i.e. lasI, lasR, rhlI, rhlR, lasA, lasB, phzA1 and rhlA) in P. aeruginosa PAO1. Naringenin also dramatically reduced the production of the acylhomoserine lactones N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL), which is driven by the lasI and rhlI gene products, respectively. In addition, using mutant strains deficient for autoinduction (ΔlasI and ΔrhlI) and LasR- and RhlR-based biosensors, it was shown that QS inhibition by naringenin not only is the consequence of a reduced production of autoinduction compounds but also results from a defect in the proper functioning of the RlhR-C4-HSL complex. Widely distributed in the plant kingdom, flavonoids are known for their numerous and determinant roles in plant physiology, plant development and in the success of plant-rhizobia interactions, but, as shown here, some of them also have a role as inhibitors of the virulence of pathogenic bacteria by interfering with QS mechanisms.

Ntann12, encoding a polypeptide homologous to annexins, was found previously to be induced upon infection of tobacco with the bacterium Rhodococcus fascians. In this study, Ntann12 is shown to bind negatively charged phospholipids in a Ca(2+)-dependent manner. In plants growing in light conditions, Ntann12 is principally expressed in roots and the corresponding protein was mainly immunolocalized in the nucleus. Ntann12 expression was inhibited following plant transfer to darkness and in plants lacking the aerial part. However, an auxin (indole-3-acetic acid) treatment restored the expression of Ntann12 in the root system in dark conditions. Conversely, polar auxin transport inhibitors such as 1-naphthylphthalamic acid (NPA) or 2,3,5-triiodobenzoic acid (TIBA) inhibited Ntann12 expression in light condition. These results indicate that the expression of Ntann12 in the root is linked to the perception of a signal in the aerial part of the plant that is transmitted to the root via polar auxin transport.

The phytopathogenic Actinomycete Rhodococcus fascians induces leafy galls on a wide range of hosts, causing major economical losses in the ornamentals industry. Although differences in the responsivity occur within species, no plant tested so far could be considered resistant to R. fascians strain D188 infection. Here, we observed that members of the genus Dalbergia, which belong to the Fabaceae, did not develop leafy galls when challenged with R. fascians and we set out to unravel the mechanism of this recalcitrance. Whereas organic extracts of Dalbergia tissues exhibited toxicity towards the bacteria, more importantly, dichloromethane bark extracts inhibited the induction of bacterial virulence gene expression without any apparent loss of viability, illustrating that resistance is likely multifactorial. The virulence quencher was identified as a new prenylated isoflavanone, termed perbergin, and specifically targeted the AttR regulon (a LysR-type transcriptional regulator) which is imperative for the switch of R. fascians from an epiphytic to a pathogenic lifestyle. The mode of action of perbergin demonstrated that just like in Gram-negative host-microbe interactions, also in Gram-positive phytopathogens autoregulation is being targeted by the plant as an efficient means of defence. Moreover, the identification of perbergin opens the path to disease control in affected nurseries., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Quorum-sensing (QS) regulates the production of key virulence factors in Pseudomonas aeruginosa and other important pathogenic bacteria. In this report, extracts of leaves and bark of Combretum albiflorum (Tul.) Jongkind (Combretaceae) were found to quench the production of QS-dependent factors in P. aeruginosa PAO1. Chromatographic fractionation of the crude active extract generated several active fractions containing flavonoids, as shown by their typical spectral features. Purification and structural characterization of one of the active compounds led to the identification of the flavan-3-ol catechin [(2R,3S)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-1(2H)-benzopyran-3,5,7-triol]. The identity of catechin as one of the active molecules was confirmed by comparing the high-pressure liquid chromatography profiles and the mass spectrometry spectra obtained for a catechin standard and for the active C. albiflorum fraction. Moreover, standard catechin had a significant negative effect on pyocyanin and elastase productions and biofilm formation, as well as on the expression of the QS-regulated genes lasB and rhlA and of the key QS regulatory genes lasI, lasR, rhlI, and rhlR. The use of RhlR- and LasR-based biosensors indicated that catechin might interfere with the perception of the QS signal N-butanoyl-l-homoserine lactone by RhlR, thereby leading to a reduction of the production of QS factors. Hence, catechin, along with other flavonoids produced by higher plants, might constitute a first line of defense against pathogenic attacks by affecting QS mechanisms and thereby virulence factor production.

RING (really interesting new gene)-H2 domain-containing proteins are widely represented in plants and play important roles in the regulation of many developmental processes as well as in plant-environment interactions. In the present report, experiments were performed to unravel the role of the poplar gene PtaRHE1, coding for a RING-H2 protein. In vitro ubiquitination assays indicate a functional E3 ligase activity for PtaRHE1 with the specific E2 ubiquitin-conjugating enzyme UbcH5a. The overexpression of PtaRHE1 in tobacco resulted in a pleiotropic phenotype characterized by a curling of the leaves, the formation of necrotic lesions on leaf blades, growth retardation, and a delay in floral transition. The plant gene expression response to PtaRHE1 overexpression provided evidence for the up-regulation of defence- and/or programmed cell death-related genes. Moreover, genes coding for WRKY transcription factors as well as for mitogen-activated protein kinases, such as wound-induced protein kinase (WIPK), were also found to be induced in the transgenic lines as compared with the wild type. In addition, histochemical beta-glucuronidase staining showed that the PtaRHE1 promoter is induced by plant pathogens and by elicitors such as salicylic acid and cellulase. Taken together, these results suggest that the E3 ligase PtaRHE1 plays a role in the ubiquitination-mediated regulation of defence response, possibly by acting upstream of WIPK and/or in the activation of WRKY factors.

The phytopathogen Rhodococcus fascians induces the development of leafy gall, which is considered to be its ecological niche. To obtain a view of the metabolic changes occurring in R. fascians during this process, an in vitro system was used where bacteria are grown in the presence of a leafy gall extract, a condition mimicking that found by the bacteria in infected plants. Proteins of R. fascians grown for 24 h under these conditions were displayed by two-dimensional polyacrylamide gel electrophoresis. Fifteen polypeptides showing a differential accumulation in response to the inducing conditions were analyzed by mass spectrometry. Two polypeptides potentially linked to the Krebs cycle, a pyruvate dehydrogenase and a fumarate hydratase, were further characterized and shown to be downregulated at the transcriptional level. The identification of these two enzymes suggests that R. fascians may shift its metabolism during the interaction with plants from the Krebs cycle to the glyoxylate shunt.

As the largest class of disease resistance R genes, the genes encoding nucleotide binding site and leucine-rich repeat proteins ("NBS-LRR genes") play a critical role in defending plants from a multitude of pathogens and pests. The diversity of NBS-LRR genes was examined in the Populus trichocarpa draft genome sequence. The NBS class of genes in this perennial tree is large and diverse, comprised of approximately 400 genes, at least twice the complement of Arabidopsis. The NBS family can be divided into multiple subfamilies with distinct domain organizations. It includes 119 Coiled-Coil-NBS-LRR genes, 64 TIR-NBS-LRR genes, 34 BED-finger-NBS-LRR, and both truncated and unusual NBS- and NBS-LRR-containing genes. The transcripts of only 34 NBS-LRR genes were detected in rust-infected and non-infected leaves using a whole-genome oligoarray. None showed an altered expression two days post inoculation.

SUMMARY Annexins are calcium-binding proteins that have been associated in plants with different biological processes such as responses to abiotic stress and early nodulation stages. Until now, the implication of annexins during plant-pathogen interactions has not been reported. Here, a novel plant annexin gene induced in tobacco BY-2 cell suspension cultures infected with the phytopathogenic bacterium Rhodococcus fascians (strain D188) has been identified. Expression of this gene, called Ntann12, is also induced, but to a lower extent, by a strain (D188-5) that is unable to induce leafy gall formation. This gene was also induced in BY-2 cells infected with Pseudomonas syringae but not in cells infected with Agrobacterium tumefaciens or Escherichia coli. Ntann12 expression was also found to be stimulated by abiotic stress, including NaCl and abscissic acid, confirming a putative role in stress signal transduction pathways. In addition, promoter-GUS analyses using homozygous transgenic tobacco seedlings showed that the developmentally controlled expression of Ntann12 is altered upon R. fascians infection. Finally, up-regulation of Ntann12 during leafy gall ontogenesis was confirmed by RT-qPCR. Discussion is focused on the potential role of Ntann12 in biotic and abiotic stress responses and in plant development, both processes that may involve Ca(2+)-dependent signalling.

* To characterize plant cell cycle activation following Rhodococcus fascians infection, bacterial impact on cell cycle progression of tobacco BY-2 cells was investigated. * S-phase-synchronized BY-2 cells were cocultivated with R. fascians and cell cycle progression was monitored by measuring mitotic index, cell cycle gene expression and flow cytometry parameters. Cell cycle alteration was further investigated by cDNA-AFLP (amplified fragment length polymorphism). * It was shown that cell cycle progression of BY-2 cells was accelerated only upon infection with bacteria whose virulence gene expression was induced by a leafy gall extract. Thirty-eight BY-2 genes showed a differential expression within 6 h post-infection. Among these, seven were previously associated with specific plant cell cycle phases (in particular S and G2/M phases). Several genes also showed a differential expression during leafy gall formation. * R. fascians-infected BY-2 cells provide a simple model to identify plant genes related to leafy gall development. R. fascians can also be regarded as a useful biotic agent to alter cell cycle progression and, thereby, gain a better understanding of cell cycle regulation in plants.

Vascular secondary growth results from the activity of the vascular cambium, which produces secondary phloem and secondary xylem. By means of cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis along aspen stems, several potential regulatory genes involved in the progressive transition from primary to secondary growth were identified. A total of 83 unique transcript-derived fragments (TDFs) was found to be differentiated between the top and the bottom of the stem. An independent RT-PCR expression analysis validated the cDNA-AFLP profiles for 19 of the TDFs. Among these, seven correspond to new genes encoding putative regulatory proteins. Emphasis was laid upon two genes encoding, respectively, an AP2/ERF-like transcription factor (PtaERF1) and a RING finger protein (PtaRHE1); their differential expression was further confirmed by reverse northern analysis. In situ RT-PCR revealed that PtaERF1 was expressed in phloem tissue and that PtaRHE1 had a pronounced expression in ray initials and their derivatives within the cambial zone. These results suggest that these genes have a potential role in vascular tissue development and/or functioning.

Lignin is a major component of wood, the most widely used raw material for the production of pulp and paper. Although the biochemistry and molecular biology underpinning lignin production are better understood than they are for the other wood components, recent work has prompted a number of re-evaluations of the lignin biosynthetic pathway. Some of the work on which these revisions have been based involved the investigation of transgenic plants with modified lignin biosynthesis. In addition to their value in elucidating the lignin biosynthetic pathway, such transgenic plants are also being produced with the aim of improving plant raw materials for pulp and paper production. This review describes how genetic engineering has yielded new insights into how the lignin biosynthetic pathway operates and demonstrates that lignin can be improved to facilitate pulping. The current technologies used to produce paper are presented in this review, followed by a discussion of the impact of lignin modification on pulp production. Fine-tuned modification of lignin content, composition, or both is now achievable and could have important economic and environmental benefits.

The lignin biosynthetic pathway has been studied for more than a century but has undergone major revisions over the past decade. Significant progress has been made in cloning new genes by genetic and combined bioinformatics and biochemistry approaches. In vitro enzymatic assays and detailed analyses of mutants and transgenic plants altered in the expression of lignin biosynthesis genes have provided a solid basis for redrawing the monolignol biosynthetic pathway, and structural analyses have shown that plant cell walls can tolerate large variations in lignin content and structure. In some cases, the potential value for agriculture of transgenic plants with modified lignin structure has been demonstrated. This review presents a current picture of monolignol biosynthesis, polymerization, and lignin structure.