Clonagem e expressão da celulase Xf-818 de Xylella Fastidiosa em Escherichia Coli

Xylella fastidiosa's genome was the first of a plant pathogen to be completely sequenced. Through comparative sequence analysis many genes were identified and, among them, several potentially involved in plant-pathogen interaction. However, the biological role of each gene should be assigned experimentally. On this regard, heterologous protein expression is a powerful tool to produce proteins from such genes, allowing their characterization. X. fastidiosa lives inside xylem vessels and eventually would degrade pit membranes from xylem cells to move radialy into the host. The identification of several putative plant cell wall degrading enzymes on X. fastidiosa genome prompted the assession of the function of such proteins. The open reading frame (ORF) Xf-818 was cloned into expression vector pET20b and E. coli cells harboring such plasmid exhibited cellulase activity. Using IPTG at 0.4 mmol L-1 with a 12 h incubation at 32°C are the best conditions to produce higher amounts of heterologous protein. The enzyme degrades cellulose confirming the endoglucanase activity of Xf-818.Xylella fastidiosa foi a primeira bactéria fitopatogênica que teve seu genoma completamente seqüenciado. A identificação de diversos genes, através de similaridade de seqüências, indicou os possíveis mecanismos de patogenicidade da bactéria. Entretanto, a determinação da função de um gene requer a confirmação experimental e, neste aspecto, a expressão heteróloga é uma poderosa ferramenta. X. fastidiosa coloniza somente o xilema das plantas hospedeiras e a identificação putativa de diversos genes semelhantes a enzimas que degradam a parede celular vegetal, estimularam o presente estudo de catacterização destas enzimas. A clonagem da ORF Xf-818 de X. fastidiosa no vetor de expressão pET20b possibilitou a produção da proteína heterologamente em E. coli. O emprego de IPTG a 0,4 mmol L-1 com 12 h a 32°C, possibilitou as melhores condições para E. coli produzir a proteína heteróloga. Clones de E. coli que expressam Xf-818, apresentam atividade celulásica, degradando eficientemente a celulose. A identificação de Xf-818 como uma endoglicanase foi assim confirmada

A representação das deputadas parlamentares na imprensa

Conferência final do projeto "Política no Feminino: Políticas de género e estratégias de visibilidade das deputadas parlamentares". Trata-se de um projeto de investigação de âmbito académico, financiado pela FCT, integrando uma equipa multidisciplinar e internacional com o objetivo de sistematizar a representatividade e o perfil das mulheres na Assembleia da República a partir de 1975 até à atualidade. Outro dos objetivos consistiu em analisar a presença das parlamentares nos media e compreender as respectivas estratégias de visibilidade no espaço público através da mediação dos jornalistas. A conferência reuniu especialistas nas diversas áreas incluídas na investigação, nomeadamente estudos dos media e do jornalismo, ciência política e sociologia da comunicação, bem como jornalistas, deputados, investigadores, docentes e estudantes destas áreas

Engineered Orange Ectopically Expressing the Arabidopsis beta-Caryophyllene Synthase Is Not Attractive to Diaphorina citri, the Vector of the Bacterial Pathogen Associated to Huanglongbing

[EN] Huanglongbing (HLB) is a destructive disease, associated with psyllid-transmitted phloem-restricted pathogenic bacteria, which is seriously endangering citriculture worldwide. It affects all citrus species and cultivars regardless of the rootstock used, and despite intensive research in the last decades, there is no effective cure to control either the bacterial species (Candidatus Liberibacter spp.) or their insect vectors (Diaphorina citri and Trioza erytreae). Currently, the best attempts to manage HLB are based on three approaches: (i) reducing the psyllid population by intensive insecticide treatments; (ii) reducing inoculum sources by removing infected trees, and (iii) using nursery-certified healthy plants for replanting. The economic losses caused by HLB (decreased fruit quality, reduced yield, and tree destruction) and the huge environmental costs of disease management seriously threaten the sustainability of the citrus industry in affected regions. Here, we have generated genetically modified sweet orange lines to constitutively emit (E)-beta-caryophyllene, a sesquiterpene repellent to D. citri, the main HLB psyllid vector. We demonstrate that this alteration in volatile emission affects behavioral responses of the psyllid in olfactometric and no-choice assays, making them repellent/less attractant to the HLB vector, opening a new alternative for possible HLB control in the field.This work was funded by the Fundo de Defesa da Citricultura (Fundecitrus), Sao Paulo Research Foundation (FAPESP, grant #2015/07011-3), and EU H2020 Innovation Action Program (grant #817526). Consent for research and field trial of genetically modified organisms was granted by the National Technical Biosafety Commission from Brazil (CTNBio) to Fundecitrus.Alquézar-García, B.; Linhares Volpe, HX.; Facchini Magnani, R.; Pedreira De Miranda, M.; Almeida Santos, M.; Vieira Marques, V.; Rodrigues De Almeida, M.... (2021). Engineered Orange Ectopically Expressing the Arabidopsis beta-Caryophyllene Synthase Is Not Attractive to Diaphorina citri, the Vector of the Bacterial Pathogen Associated to Huanglongbing. Frontiers in Plant Science. 12:1-15. https://doi.org/10.3389/fpls.2021.6414571151

Partial characterization of elicitors of phytoalexins in sorghum (Sorghum bicolor) obtained from Saccharomyces cerevisiae

Em sorgo são sintetizadas quatro fitoalexinas do tipo deoxiantocianidinas em resposta a tentativa de penetração fúngica e ao tratamento com elicitores. A levedura Saccharomyces cerevisiae estimula o acúmulo de fitoalexinas e tem potencial para ser utilizada como agente de controle alternativo no tratamento de doenças fúngicas em sorgo. Descrevemos aqui metodologia para a obtenção de elicitores glicoproteicos a partir da levedura S. cerevisiae, os quais estimulam o acúmulo de fitoalexinas em mesocótilos de sorgo. As frações purificadas são resistentes a autoclavagem, solúveis em etanol 50 %, ligam-se a resina aniônica DEAE-Celulose e o tratamento com proteinase reduz a atividade elicitora. Além das moléculas elicitoras, foram fracionadas moléculas que possuem a capacidade de suprimir o acúmulo de fitoalexinas pelas moléculas elicitoras. Com base nos resultados apresentados, sugere-se que moléculas possivelmente de natureza glicoproteica, oriundas da parece celular da levedura, atuem como elicitores de fitoalexinas em sorgo. Ressalta-se que da mesma forma que poucas fitoalexinas foram estudadas em monocotiledôneas, poucos são os elicitores caracterizados a apresentar atividade entre as monocotiledôneas, especialmente nas Poaceae.Sorghum produces a complex of pigmented 3- deoxyanthocyanidin phytoalexins in response to either attempted fungal penetration or elicitor treatment. The yeast Saccharomyces cerevisiae e elicits phytoalexin accumulation and is a potential biological agent for control of fungal diseases in sorghum. The present investigation establishes a procedure to purify elicitor molecules from cells of S. cerevisiae, which cause phytoalexin accumulation in sorghum mesocotyls. In addition to elicitor molecules, suppressor molecules of the same response were fractionated. The elicitor molecules are stable after autoclaving, soluble in ethanol 50 %, adsorb to the anion exchanger DEAE-Cellulose and proteinase treatment reduces their activities. These elicitors molecules, probably glycoproteins from S. cerevisiae cell walls, are one of the few to exhibit elicitor activity in monocotyledons, specially on Poaceae

Partial characterization of elicitors of phytoalexins in sorghum (Sorghum bicolor) obtained from Saccharomyces cerevisiae

Em sorgo são sintetizadas quatro fitoalexinas do tipo deoxiantocianidinas em resposta a tentativa de penetração fúngica e ao tratamento com elicitores. A levedura Saccharomyces cerevisiae estimula o acúmulo de fitoalexinas e tem potencial para ser utilizada como agente de controle alternativo no tratamento de doenças fúngicas em sorgo. Descrevemos aqui metodologia para a obtenção de elicitores glicoproteicos a partir da levedura S. cerevisiae, os quais estimulam o acúmulo de fitoalexinas em mesocótilos de sorgo. As frações purificadas são resistentes a autoclavagem, solúveis em etanol 50 %, ligam-se a resina aniônica DEAE-Celulose e o tratamento com proteinase reduz a atividade elicitora. Além das moléculas elicitoras, foram fracionadas moléculas que possuem a capacidade de suprimir o acúmulo de fitoalexinas pelas moléculas elicitoras. Com base nos resultados apresentados, sugere-se que moléculas possivelmente de natureza glicoproteica, oriundas da parece celular da levedura, atuem como elicitores de fitoalexinas em sorgo. Ressalta-se que da mesma forma que poucas fitoalexinas foram estudadas em monocotiledôneas, poucos são os elicitores caracterizados a apresentar atividade entre as monocotiledôneas, especialmente nas Poaceae.Sorghum produces a complex of pigmented 3- deoxyanthocyanidin phytoalexins in response to either attempted fungal penetration or elicitor treatment. The yeast Saccharomyces cerevisiae e elicits phytoalexin accumulation and is a potential biological agent for control of fungal diseases in sorghum. The present investigation establishes a procedure to purify elicitor molecules from cells of S. cerevisiae, which cause phytoalexin accumulation in sorghum mesocotyls. In addition to elicitor molecules, suppressor molecules of the same response were fractionated. The elicitor molecules are stable after autoclaving, soluble in ethanol 50 %, adsorb to the anion exchanger DEAE-Cellulose and proteinase treatment reduces their activities. These elicitors molecules, probably glycoproteins from S. cerevisiae cell walls, are one of the few to exhibit elicitor activity in monocotyledons, specially on Poaceae

Enzymatic characterization of endoglucanases XF810, XF818 and XF2708 from Xylella fastidiosa and purification of protein XF818 expressed in Escherichia coli.

A bactéria Xylella fastidiosa causa a clorose variegada dos citros, também conhecida como amarelinho. Em 2000, foi concluído o seqüenciamento do genoma deste organismo, o primeiro de uma bactéria fitopatogênica, um fato que estimulou o estudo dos possíveis mecanismos de patogenicidade empregados pela bactéria para causar a doença em citros. X. fastidiosa é uma bactéria limitada ao xilema, sendo transmitida de planta a planta através de insetos vetores. Especula-se que a bactéria produza enzimas que degradem a membrana da pontuação, permitindo a colonização de novos vasos do xilema. A identificação de genes com similaridade de seqüência a genes de celulases, xilanases, pectinases e proteases, fomentou o presente estudo visando caracterizar os genes Xf - 810, Xf - 818 e Xf - 2708, similares a endoglicanases. Estes genes foram clonados em vetores de expressão e as respectivas proteínas foram produzidas em Escherichia coli. Através de ensaios enzimáticos as proteínas foram caracterizadas como endoglicanases (EC 3.2.1.4), que são celulases com mecanismo endoglicolítico de ataque às moléculas de celulose. Estas celulases hidrolisam carboximetil celulose, Avicel e xilana, enquanto as enzimas Xf - 810 e Xf - 818 hidrolisam a celulose amolecida com ácido. Estas celulases degradam mais eficientemente a carboximetil celulose em pH ácido (entre 5,2 e 5,6) e na temperatura de 65 °C. Coletivamente, estas celulases são capazes de degradar polímeros solúveis e insolúveis, enquanto a enzima Xf - 818, é capaz de degradar oligossacarídeos derivados da celulose, como celotetraose e celopentaose, apresentando ampla variação catalítica. Esta celulase possui capacidade de ligação à celulose microcristalina, denotando a funcionalidade de seu domínio ligador de celulose. Desenvolvemos um protocolo, empregando cromatografia de troca aniônica, afinidade por metal (níquel) e filtração em gel, eficiente na purificação da proteína Xf - 818 expressa heterologamente em E. coli com fusão hexahistidina à extremidade N-terminal. A caracterização enzimática destas proteínas, com a confirmação da atividade celulásica, fornece subsídios para uma eventual função das celulases durante a colonização do hospedeiro pela bactéria, pois são cataliticamente funcionais. Ademais, corrobora a similaridade destes genes, verificada durante o seqüenciamento do genoma de X. fastidiosa.Xylella fastidiosa is the causal agent of citrus variegated chlorosis, also known as "amarelinho". The recent sequencing of its genome, achieved in 2000, was the first of a plant pathogen, a fact that stimulated the search for putative pathogenicity factors employed by this bacterium while infecting citrus trees. X. fastidiosa inhabits exclusively the xylem vessels, being transmitted by sharpshooter vectors. Several authors argue that the bacterium produces enzymes to degrade plant cell, as a way to colonize new xylem vessels through pit membrane degradation. The identification of putative cellulases, xylanases, pectinases and proteases on X. fastidiosa genome, led us to carry out the present work to characterize the putative products of the endoglucanase genes Xf - 810, Xf - 818 and Xf - 2708. These genes were cloned into expression vectors and the proteins were produced in Escherichia coli. Based upon enzymatic assays, those proteins were characterized as endoglucanases (EC 3.2.1.4), which are cellulases able to promote the endo-hydrolysis of cellulose chains. These cellulases degraded carboxymethylcellulose, Avicel and xylan, while only Xf - 810 and Xf - 818 degraded acid swollen cellulose. The hydrolysis of carboxymethylcellulose was higher at acidic pH between 5.2 and 5.6) and at a temperature of 65 °C. As a group, these enzymes were able to degrade soluble and insoluble cellulose derivatives, while only the cellulase Xf - 818 could hydrolyze the cello-oligosaccharides cellotetrose and cellopentose, thus showing a high catalytic diversity. This protein also has the capacity to bind microcristalline cellulose, confirming the presence of a functional cellulose-binding domain. We set a protocol, employing anion exchange, metal affinity and gel filtration chromatography, to purify the Xf - 818 enzyme expressed in E. coli as a N-hexahistine fusion tag. The endoglucanase activity studied gives support for an eventual role of such cellulases during host colonization by the bacterium. Besides that, it agrees with similarities searches observed on the sequencing of X. fastidiosa genome

Drench Application of Systemic Insecticides Disrupts Probing Behavior of Diaphorina citri (Hemiptera: Liviidae) and Inoculation of Candidatus Liberibacter asiaticus

© 2020 by the authorsCandidatus Liberibacter asiaticus (CLas) is a phloem-limited bacterium that is associated with the Huanglongbing (HLB) disease of citrus and transmitted by the psyllid, Diaphorina citri. There are no curative methods to control HLB and the prevention of new infections is essential for HLB management. Therefore, the objective of our study was to determine the effects of systemic insecticides, such as the neonicotinoids imidacloprid, thiamethoxam, and a mixture of thiamethoxam and chlorantraniliprole (diamide) on the probing behavior of CLas-infected D. citri and their effect on CLas transmission. The electrical penetration graph (EPG-DC) technique was used to monitor the stylet penetration activities of CLas-infected D. citri on sweet orange [Citrus sinensis (L.) Osbeck] ‘Valencia’ treated with systemic insecticides. Systemic insecticides disrupted the probing behavior of CLas-infected D. citri, in a way that affected CLas transmission efficiency, particularly by negatively affecting the stylet activities related to the phloem phase. All insecticides reduced (by 57–73%) the proportion of psyllids that exhibited sustainable phloem ingestion (waveform E2 > 10 min), with significant differences observed on plants treated with thiamethoxam and thiamethoxam + chlorantraniliprole. The transmission rate of CLas with high inoculum pressure (five CLas-infected D. citri per plant and a seven-day inoculation access period) to untreated control plants was 93%. In contrast, CLas transmission was reduced to 38.8% when test plants were protected by systemic insecticides. Our results indicated that all insecticides tested presented a potential to reduce CLas inoculation by an average of 59%; therefore, these insecticides can be used to reduce the spread of HLB.Financial support was received from National Council for Scientific and Technological Development (CNPq) and supported by Fund for Citrus Protection (Fundecitrus). First author received a Posdoc scholarship from CNPq/Brazil (300581/2016-5); the last author received a fellowship CNPq/Brazil (Proc. 301805/2018-0).Peer reviewe

The Genome of "Candidatus Liberibacter asiaticus " Is Highly Transcribed When Infecting the Gut of Diaphorina citri

[EN] The Asian citrus psyllid, Diaphorina citri, is the vector of the bacterium "Candidatus Liberibacter asiaticus" (Las), associated with the devastating, worldwide citrus disease huanglongbing. In order to explore the molecular interactions of this bacterium with D. citri during the vector acquisition process, cDNA libraries were sequenced on an Illumina platform, obtained from the gut of adult psyllids confined in healthy (H) and in Las-infected young shoots (Las) for different periods of times (I = 1/2 days, II = 3/4 days, and III = 5/6 days). In each sampling time, three biological replicates were collected, containing 100 guts each, totaling 18 libraries depleted in ribosomal RNA. Reads were quality-filtered and mapped against the Chinese JXGC Las strain and the Floridian strain UF506 for the analysis of the activity of Las genome and SC1, SC2, and type 3 (P-JXGC-3) prophages of the studied Las strain. Gene activity was considered only if reads of at least two replicates for each acquisition access period mapped against the selected genomes, which resulted in coverages of 44.4, 79.9, and 94.5% of the JXGC predicted coding sequences in Las I, Las II, and Las III, respectively. These genes indicate an active metabolism and increased expression according to the feeding time in the following functional categories: energy production, amino acid metabolism, signal translation, cell wall, and replication and repair of genetic material. Pilins were among the most highly expressed genes regardless of the acquisition time, while only a few genes from cluster I of flagella were not expressed. Furthermore, the prophage region had a greater coverage of reads for SC1 and P-JXGC-3 prophages and low coverage in SC2 and no indication of activity for the lysis cycle. This research presents the first descriptive analysis of Las transcriptome in the initial steps of the D. citri gut colonization, where 95% of Las genes were active.Financial support from FAPESP (2015/07011-3) and postdoctoral fellowship to FB (FAPESP 2018/24234-4). Doctorate scholarship to JD from CNPq (141042/2017-6).Darolt, JC.; Bento, FDMM.; Merlin, BL.; Peña, L.; Consoli, FL.; Wulff, NA. (2021). The Genome of "Candidatus Liberibacter asiaticus " Is Highly Transcribed When Infecting the Gut of Diaphorina citri. Frontiers in Microbiology. 12. https://doi.org/10.3389/fmicb.2021.6877251

Citrus sudden death-associated virus (CSDaV) and citrus tristeza virus (CTV) in eleven rootstocks for ‘Valência’ sweet orange

Abstract Citrus sudden death (CSD) is a highly destructive disease and has caused the eradication of millions of trees in southern Brazil within the last 15 years. In spite of the exact cause of CSD has not been determined, evidences have shown that this disease can be transmitted by biotic vectors. Disease incidence in sweet orange scions is related to the rootstock, and the combination with ‘Rangpur’ lime is the most affected. On the other hand, there are evidences of a relation between CSD affected trees and the presence of the Citrus sudden death associated virus (CSDaV) and/or Citrus tristeza virus (CTV). Based on such information, this study has been carried out to determine the presence of CSDaV and CTV, and the association between each other in eleven rootstocks for ‘Valencia’ sweet orange. The results presented herein showed differences related to the presence of CSDaV and CTV in different rootstocks for ‘Valencia’ sweet orange and no relation between the presence of CSDaV and CTV

Molecular Characterization and Detection of 16SrIII Group Phytoplasma Associated with Huanglongbing Symptoms.

When huanglongbing (HLB) was found in Brazil in 2004, 'Candidatus Liberibacter americanus' was infecting most of the trees while 'Ca. L. asiaticus' was present in a minor proportion. Currently, 'Ca. L. asiaticus' is the predominant bacterium associated with HLB in citrus trees in São Paulo (SP) and Minas Gerais (MG) States, the major citrus-growing regions in Brazil. A phytoplasma from the 16SrIX group was associated with HLB symptoms in Brazil in 2007, in plants free of Liberibacter spp. In this report, HLB samples testing negative for 'Ca. L. asiaticus', 'Ca. L. americanus', and 16SrIX phytoplasma were infected with 16SrIII phytoplasmas. Coinfection with 'Ca. L. asiaticus' and 16SrIII was also found. The 16S ribosomal RNA (rRNA) gene sequences from 22 samples were obtained and sequenced, confirming that the 16SrIII group phytoplasma is associated with HLB symptoms in SP and MG States. Ten single-nucleotide polymorphisms (SNPs) were found in the 1,427-bp 16S rRNA gene sequences from 16SrIII phytoplasmas from citrus, whereas none was detected in 16S rRNA gene sequences among 16SrIX phytoplasma from citrus. Ribosomal protein (rp) rpsSrplVrpsC gene sequences were amplified with 16SrIII group-specific primers, sequenced from a subset of nine samples, and assembled into three groups based on eight SNPs. SNPs in 16S rRNA gene and rp gene sequences are common in 16SrIII phytoplasmas from other hosts and this phytoplasma group is widespread in South America. 16SrIII phytoplasmas highly related are commonly found in Melia azedarach, a widespread tree in Brazil and Argentina. The finding of a new phytoplasma associated with HLB symptoms belonging to the 16SrIII group reinforces the need to develop diagnostic tools to assess HLB-associated microbiomes