Type III secretion inhibitors for the management of bacterial plant diseases

The identification of chemical compounds that prevent and combat bacterial diseases is fundamental for crop production. Bacterial virulence inhibitors are a promising alternative to classical control treatments, because they have a low environmental impact and are less likely to generate bacterial resistance. The major virulence determinant of most animal and plant bacterial pathogens is the type III secretion system (T3SS). In this work, we screened nine plant extracts and 12 isolated compounds including molecules effective against human pathogens for their capacity to inhibit the T3SS of plant pathogens and for their applicability as virulence inhibitors for crop protection. The screen was performed using a luminescent reporter system developed in the model pathogenic bacterium Ralstonia solanacearum. Five synthetic molecules, one natural product and two plant extracts were found to down‐regulate T3SS transcription, most through the inhibition of the regulator hrpB. In addition, for three of the molecules, corresponding to salicylidene acylhydrazide derivatives, the inhibitory effect caused a dramatic decrease in the secretion capacity, which was translated into impaired plant responses. These candidate virulence inhibitors were then tested for their ability to protect plants. We demonstrated that salicylidene acylhydrazides can limit R. solanacearum multiplication in planta and protect tomato plants from bacterial speck caused by Pseudomonas syringae pv. tomato. Our work validates the efficiency of transcription reporters to discover compounds or natural product extracts that can be potentially applied to prevent bacterial plant diseases

Complete genome sequence of the potato pathogen Ralstonia solanacearum UY031

Ralstonia solanacearum is the causative agent of bacterial wilt of potato. Ralstonia solanacearum strain UY031 belongs to the American phylotype IIB, sequevar 1, also classified as race 3 biovar 2. Here we report the completely sequenced genome of this strain, the first complete genome for phylotype IIB, sequevar 1, and the fourth for the R. solanacearum species complex. In addition to standard genome annotation, we have carried out a curated annotation of type III effector genes, an important pathogenicity-related class of genes for this organism. We identified 60 effector genes, and observed that this effector repertoire is distinct when compared to those from other phylotype IIB strains. Eleven of the effectors appear to be nonfunctional due to disruptive mutations. We also report a methylome analysis of this genome, the first for a R. solanacearum strain. This analysis helped us note the presence of a toxin gene within a region of probable phage origin, raising the hypothesis that this gene may play a role in this strain's virulence

Comparative analysis of Ralstonia solanacearum methylomes

Ralstonia solanacearum is an important soil-borne plant pathogen with broad geographical distribution and the ability to cause wilt disease in many agriculturally important crops. Genome sequencing of multiple R. solanacearum strains has identified both unique and shared genetic traits influencing their evolution and ability to colonize plant hosts. Previous research has shown that DNA methylation can drive speciation and modulate virulence in bacteria, but the impact of epigenetic modifications on the diversification and pathogenesis of R. solanacearum is unknown. Sequencing of R. solanacearum strains GMI1000 and UY031 using Single Molecule Real-Time technology allowed us to perform a comparative analysis of R. solanacearum methylomes. Our analysis identified a novel methylation motif associated with a DNA methylase that is conserved in all complete Ralstonia spp. genomes and across the Burkholderiaceae, as well as a methylation motif associated to a phage-borne methylase unique to R. solanacearum UY031. Comparative analysis of the conserved methylation motif revealed that it is most prevalent in gene promoter regions, where it displays a high degree of conservation detectable through phylogenetic footprinting. Analysis of hyper- and hypo-methylated loci identified several genes involved in global and virulence regulatory functions whose expression may be modulated by DNA methylation. Analysis of genome-wide modification patterns identified a significant correlation between DNA modification and transposase genes in R. solanacearum UY031, driven by the presence of a high copy number of ISrso3 insertion sequences in this genome and pointing to a novel mechanism for regulation of transposition. These results set a firm foundation for experimental investigations into the role of DNA methylation in R. solanacearum evolution and its adaptation to different plants

Control strategies and gene expression dynamics of the plant pathogen Ralstonia solanacearum = Estratègies de control i dinàmica d'expressió gènica en el fitopatogen Ralstonia solanacearum

[eng] The plant pathogen Ralstonia solanacearum is the causal agent of bacterial wilt, a highly aggressive disease responsible for important worldwide economic losses. Many virulence factors in R. solanacearum have been already identified; however, their transcriptional regulation during disease development remained unknown. In an effort to better characterize the gene expression changes driving bacterial virulence, we first provided the complete genome sequence of the potato R. solanacearum UY031 strain as a tool to perform robust transcriptomics in planta. By taking advantage of the novel sequencing technology called SMRT, we also supplied hints on the methylome profile and its contribution to virulence gene expression in UY031. In this work, we performed two different in planta transcriptome approaches at different potato infection stages. On one hand, we analyzed the bacterial gene expression during root colonization and demonstrated that, although it is cost-ineffective, microbial transcriptomes in planta at low bacterial densities are possible without a prior enrichment of prokaryotic RNA. Furthermore, we identified a novel player controlling bacterial fitness during early infection stages that we named RepR for Repressor Regulator, since we discovered that it is a repressor of specific metabolic pathways. On the other hand, we performed a time- course transcriptome and show that expression of R. solanacearum virulence factors and metabolism is dynamic along the infection process. With our system, we validated the expression patterns of known virulence factors such as the Type III Secretion System (T3SS) or the flagellum, and unraveled the profiles of others like Type IVb pili or the T6SS. Contrary to the assumption that the T3SS might play only a role at early infection stages, we demonstrate that effector transcription is extremely high in advanced disease stages. Finally, we performed a pilot test to identify T3SS inhibitors and demonstrate that some salicylidene acylhydrazides can potentially prevent bacterial plant diseases via T3SS transcription inhibition. This work adds growing knowledge on the pathogen behavior and its physiology at different points of the disease, which could eventually lead to the identification of new drugs targeting keys steps in disease development.[cat] alstonia solanacearum és l’agent causant del marciment bacterià en plantes, una malaltia altament agressiva i responsable de considerables pèrdues econòmiques d’impacte mundial. Molts factors de virulència de R. solanacearum han sigut identificats, però la seva regulació transcripcional al llarg del desenvolupament de la malaltia encara es desconeixia. En un intent de caracteritzar els canvis en l’expressió genètica que modulen la virulència del bacteri, en primer lloc hem proporcionat la seqüència completa del genoma de la soca de patateres R. solanacearum UY031 com a eina per a dur a terme transcriptomes robustos dins de la planta. Gràcies a la nova tecnologia de seqüenciació anomenada SMRT, també proporcionem algunes pistes sobre el seu perfil de metilació i la contribució d’aquest en l’expressió de gens de virulència a UY031. En aquest estudi hem realitzat dos transcriptomes del bacteri en patateres en diferents estadis d’infecció. Per una banda hem analitzat l’expressió genètica bacteriana durant la colonització de l’arrel i hem demostrat que, malgrat ser poc rentable, és possible analitzar el transcriptoma del bacteri dins de la planta sense enriquir prèviament les mostres amb ARN procariota. Així mateix, hem identificat un nou membre que regula l’eficàcia biològica del bacteri durant els estadis inicials de la infecció que hem anomenat RepR, de Repressor Regulador, ja que hem descobert que reprimeix rutes metabòliques concretes. Per altra banda, hem fet un transcriptoma a diferents estadis de la infecció i demostrem que l’expressió de factors de virulència i del metabolisme de R. solanacearum és dinàmica al llarg del procés infectiu. Amb el nostre sistema, hem validat els patrons d’expressió de factors de virulència ja coneguts, com el Sistema de Secreció de Tipus III (SST3) o el flagel, i hem desxifrat els perfils d’altres factors com el dels pilus de tipus IVb o el SST6. En contra de l’assumpció que el SST3 juga principalment un paper als estadis primerencs de la infecció, hem demostrat que la transcripció de molts efectors és extremadament alta en estadis avançats de la malaltia. Finalment, hem dut a terme una prova pilot per a identificar inhibidors del SST3 i hem demostrat que algunes salicidèn-acilhidrazides tenen potencial per a prevenir malalties bacterianes de plantes mitjançant la inhibició de la transcripció del SST3. Aquest treball afegeix nou coneixement en el comportament i la fisiologia del patogen en diferents estadis de la malaltia, que amb el temps podria contribuir a la identificació de nous fàrmacs dirigits en passos claus en el desenvolupament de la malaltia

Les lleis de la dinàmica

Unitat didàctica del curs de GES 2 (especialitat Ciències Naturals). El·laborada dins el Màster de Formació del Professorat d'Educació Secundària i Batxillerat UPF-UOCTutor: Marcel CostaMentora: Eulàlia Roger (CFA L'Alzina, Escola de formació d'adults de Cerdanyola del Vallès)Aquesta Unitat Didàctica sobre les Lleis de la Dinàmica s’ha dissenyat com a part del mòdul de Física i Química II del GES2 (segon nivell del Graduat d’Educació Secundària per a adults). El contingut està plantejat per a dur-se a terme en 6 sessions d’una hora i mitja cadascuna. Cada sessió consta de diverses situacions d’aprenentatge familiars i conegudes pels alumnes, sobre qüestions i fenòmens de la vida quotidiana. Les primeres 4 sessions giren al voltant d’un experiment a l’aula i cadascun es desenvolupa en un moment diferent de la sessió i correspon a una etapa del cicle d’aprenentatge. El treball sobre els experiments inclouen l’observació, formulació d’hipòtesis, presa i anàlisi de dades i extrapolació a altres situacions. La penúltima sessió està dedicada a repassar els principals conceptes i teories. L’última sessió es destina a la prova final individual, la qual és co-avaluada després pels alumnes

Les lleis de la dinàmica

Unitat didàctica del curs de GES 2 (especialitat Ciències Naturals). El·laborada dins el Màster de Formació del Professorat d'Educació Secundària i Batxillerat UPF-UOCTutor: Marcel CostaMentora: Eulàlia Roger (CFA L'Alzina, Escola de formació d'adults de Cerdanyola del Vallès)Aquesta Unitat Didàctica sobre les Lleis de la Dinàmica s’ha dissenyat com a part del mòdul de Física i Química II del GES2 (segon nivell del Graduat d’Educació Secundària per a adults). El contingut està plantejat per a dur-se a terme en 6 sessions d’una hora i mitja cadascuna. Cada sessió consta de diverses situacions d’aprenentatge familiars i conegudes pels alumnes, sobre qüestions i fenòmens de la vida quotidiana. Les primeres 4 sessions giren al voltant d’un experiment a l’aula i cadascun es desenvolupa en un moment diferent de la sessió i correspon a una etapa del cicle d’aprenentatge. El treball sobre els experiments inclouen l’observació, formulació d’hipòtesis, presa i anàlisi de dades i extrapolació a altres situacions. La penúltima sessió està dedicada a repassar els principals conceptes i teories. L’última sessió es destina a la prova final individual, la qual és co-avaluada després pels alumnes

Novel plant inputs influencing Ralstonia solanacearum during infection

Ralstonia solanacearum is a soil and water-borne pathogen that can infect a wide range of plants and cause the devastating bacterial wilt disease. To successfully colonize a host, R. solanacearum requires the type III secretion system (T3SS), which delivers bacterial effector proteins inside the plant cells. HrpG is a central transcriptional regulator that drives the expression of the T3SS and other virulence determinants. hrpG transcription is highly induced upon plant cell contact and its product is also post-transcriptionally activated by metabolic signals present when bacteria are grown in minimal medium (MM). Here, we describe a transcriptional induction of hrpG at early stages of bacterial co-culture with plant cells that caused overexpression of the downstream T3SS effector genes. This induction was maintained in a strain devoid of prhA, the outer membrane receptor that senses bacterial contact with plant cells, demonstrating that this is a response to an unknown signal. Induction was unaffected after disruption of the known R. solanacearum pathogenicity regulators, indicating that it is controlled by a non-described system. Moreover, plant contact-independent signals are also important in planta, as shown by the hrpG induction triggered by apoplastic and xylem extracts. We also found that none of the amino acids or sugars present in the apoplast and xylem saps studied correlated with hrpG induction. This suggests that a small molecule or an environmental condition is responsible for the T3SS gene expression inside the plants. Our results also highlight the abundance and diversity of possible carbon, nitrogen and energy sources likely used by R. solanacearum during growth in planta