Assessment of resistance of hybrids and lineages of passion fruit to bacteriosis under protected cultivation / Avaliação da resistência de híbridos e linhagens de maracujazeiro à bacteriose sob cultivo protegido

Yield and longevity of yellow passion fruit have been reduced by diseases such as the bacterial spot caused by Xanthomonas axonopodis pv. passiflorae. Genetic resistance has been confirmed as the most efficient and economical correct option to minimize this disease problem. Aiming at it, the objective of this research was to evaluate the incidence, severity and progression of the disease in 12 genotypes of sour passion fruit, in seedling stage in nursery greenhouse after inoculation of Xanthomonas axonopodis pv. passiflorae. The inoculation was performed with an isolate collected in the Pipiripau Rural Nucleus, Brasilia-DF, named UnB-1397 (1x106 CFU/mL), through induction of injuries. There were performed four assessments, with interval of 7 days except the first which was performed 11 days after inoculation. The incidence was estimated by the percentage of plants affected. To evaluate the severity, it was used the diagrammatic scale validated by Costa et al. (2018), with adaptations, using the measurement of the affected area by necrotic lesions on the leaf. All genotypes were susceptible to bacteriosis, 5 being considered moderately susceptible: F1BRS Pérola do Cerrado x Rosa Intenso, Mar20#21, Mar20#15b, Mar20#24xMar20#40 and FB200PL4R2 x Mar20#2005, with a mean of severity ranging from 11 to 25% of injured area in leaves.

A produtividade e a longevidade dos pomares de maracujazeiro-azedo têm sido comprometidas em razão de doenças como a bacteriose, causada por Xanthomonas axonopodis pv. passiflorae. A resistência genética tem se confirmado como a opção mais eficiente e econômica para minimizar tal problema. Dessa forma, o objetivo deste trabalho foi avaliar a reação de 12 genótipos de maracujazeiro, em fase de mudas, sob cultivo protegido, à Xanthomonas axonopodis pv. passiflorae. A inoculação com isolado denominado UnB-1397 (1x106 CFU/mL),coletado no Núcleo Rural de Pipiripau, Brasília-DF, se deu pela indução de ferimentos. Foram realizadas 4 avaliações, com intervalo de 7 dias, sendo a primeira avaliação realizada 11 dias após a inoculação. A incidência foi estimada pela porcentagem de plantas afetadas. Para avaliação da severidade, foi utilizada escala diagramática validada por Costa et al. (2018), com adaptações, utilizando-se a mensuração da área foliar atingida por lesões nas folhas. Todos os genótipos se mostraram suscetíveis à bacteriose, sendo 5 considerados moderadamente suscetíveis: F1 BRS Pérola do Cerrado x Rosa Intenso, Mar20#21, Mar20#15b, Mar20#24 x Mar20#40 e FB200PL4R2 x Mar20#2005, apresentando uma média de severidade que variou de 11 a 25% de área ou tecidos foliares lesionados.

Reaction of yellow passion fruit to passion fruit woodiness disease and to bacterial spot / Reação de maracujazeiro azedo à virose do endurecimento dos frutos e à bacteriose

The use of resistant varieties is a promising strategy for passion fruit woodiness disease (PWD) (Cowpea aphid-borne mosaic virus - CABMV) and bacterial spot (Xanthomonas axonopodis pv. passiflorae - Xap) control in yellow passion fruit (Passiflora edulis Sims). This study aimed at evaluating the reaction of nine genotypes of yellow passion fruit to both mechanically inoculated CABMV and Xap, under protected cultivation. The experiment was conducted using a randomized block design with subdivided parcels comprised of nine treatments, four repetitions, six replications per genotype, and five evaluations. Disease incidence (% plants infected) and severity (% of total leaf area with necrotic lesions or leaf symptoms) were calculated at 7-day intervals. All genotypes evaluated were classified as moderately susceptible to PWD. MAR20#10, MAR20#41, and Rosa Intenso were classified as moderately resistant to bacterial spot whereas the remaining genotypes were classified as moderately susceptible. Genotypes Rosa Intenso, MAR20#41, MAR20#15, and MSCA stood out for presenting the lowest PWD mean severity scores as well as the greatest numbers of plants presenting resistance to PWD after five evaluations. MAR20#10, MAR20#41, and Rosa Intenso demonstrated the lowest bacterial spot mean severity scores. Among the genotypes selected, Rosa Intenso and MAR20#41 were the most promising genotypes for presenting the lowest severity scores for both PWD and bacterial spot disease.

O uso de variedades resistentes é uma estratégia promissora para o controle da virose do endurecimento dos frutos (VEF) (Cowpea aphid-borne mosaic virus - CABMV) e da bacteriose (Xanthomonas axonopodis pv. passiflorae - Xap) no maracujazeiro amarelo (Passiflora edulis Sims). Este estudo objetivou avaliar a reação de nove genótipos de maracujazeiro amarelo à CABMV e Xap, ambos inoculados mecanicamente, sob cultivo protegido. O experimento foi conduzido em delineamento de blocos casualizados com parcelas subdivididas, composto por nove tratamentos, quatro repetições, seis plantas por genótipo e cinco avaliações. A incidência (% plantas infectadas) e a severidade (% da área foliar total com lesões necróticas ou sintomas foliares) das doenças foram calculadas em intervalos de sete dias. Todos os genótipos avaliados foram classificados como moderadamente suscetíveis à VEF. MAR20#10, MAR20#41 e Rosa Intenso foram classificados como moderadamente resistente à bacteriose enquanto os demais genótipos foram classificados como moderadamente suscetíveis. Os genótipos Rosa Intenso, MAR20#41, MAR20#15 e MSCA se destacaram por apresentarem menores severidades médias da VEF bem como pelo maior número de plantas apresentando resistência à virose após as cinco avaliações. MAR20#10, MAR20#41 e Rosa Intenso demonstraram as menores severidades médias de bacteriose. Entre os genótipos selecionados, Rosa Intenso e MAR20#41 foram os mais promissores por apresentarem os menores valores de severidade para a VEF e para a bacteriose.

Yield and rheological properties of exopolysaccharide from a local isolate: Xanthomonas axonopodis pv. vesicatoria

Background: The aim of the present study was to evaluate gum productivity of a local strain, Xanthomonas axonopodis pv. vesicatoria, isolated from pepper plant, and its rheological behavior for the first time compared to the standard strain, Xanthomonas campestris DSM 19000 (NRRL B-1459). The influence of operational conditions (agitation rate and inoculum volume) on gum production and rheological properties of gums from the Xanthomonas strains were investigated. Results: The isolated strain of Xanthomonas showed similar xanthan yield compared to the standard strain. Furthermore, this study clearly confirmed that gum yield depended on bacterial strain, agitation rate, and inoculum size. The most suitable conditions for the gum production in an orbital shaker in terms of agitation rate and inoculum size were 180 rpm and 5%, respectively, resulting in an average production of 10.96 and 11.19 g/L for X. axonopodis pv.vesicatoria and X. campestris DSM 19000, respectively. Regarding the rheological properties, Ostwald-de-Waele and power law models were used to describe flow and oscillatory behavior of the gum solutions, respectively. Consistency of the novel gum solution remarkably was much higher than the commercial xanthan gum solution. Flow and oscillatory behavior and their temperature ramps showed that weak gel-like structure could be obtained with less gum concentrations when the novel gum was used. Conclusion: Therefore, yield and technological properties of the aqueous solutions of the exopolysaccharide synthesized by X. axonopodis pv. vesicatoria were observed to be more suitable for industrial production.

Xanthomonas fuscans subsp. fuscans causing wilt symptoms in bean plants (Phaseolus vulgaris) in Brazil / Xanthomonas fuscans subsp. fuscans causando sintomas de murcha em feijoeiro (Phaseolus vulgaris) no Brasil

Common and fuscous blights of bean are diseases widely distributed in the world. The most commonly observed symptoms are spots on leaves, stems, pods and seeds. In December 2009, bean plants cv. Uirapuru showing symptoms of wilt similar to those induced by Curtobacterium flaccumfaciens pv. flaccumfaciens were observed in a commercial crop located in the county of Itararé, State of São Paulo, Brazil. The plants were at the last cycle stage with mature pods and these symptoms were noted in the majority of the growing area. Optical microscopic observations of discolored vascular tissue from diseased stems revealed the presence of bacterial masses oozed from infected tissue, indicating that the disease was caused by bacterial pathogen. Isolations on nutrient agar showed circular, convex, yellow colonies with smooth edges. The causal bacterium was Gramnegative and produced a dark brown pigment in culture medium. Biochemical, cultural and physiological tests confirmed its identity as Xanthomonas fuscans subsp. fuscans(syn. Xanthomonas campestris pv. phaseoli"var. fuscans"). The pathogenicity of the isolates was confirmed by artificial inoculations. Systemic infection has been reported in the literature but these kinds of symptoms are not currently observed in Brazilian fields. Bacterial strains were deposited on the Phytobacteria Culture Collection of Instituto Biológico (IBSBF - www.biologico.sp.gov.br/bacterias/php) under accession numbers 2813 and 3028.(AU)

O crestamento bacteriano comum do feijoeiro é uma doença amplamente distribuída no mundo e os sintomas comumente observados são manchas nas folhas, hastes, vagens e sementes. Em dezembro de 2009, plantas de feijoeiro cv. Uirapuru com sintomas de murcha similares aos produzidos por Curtobacterium flaccumfaciens pv. flaccumfaciens foram observadas em campos comerciais localizados no município de Itararé, estado de São Paulo, Brasil. As plantas encontravam-se no final do ciclo, com as vagens já formadas. Os sintomas foram notados em quase a totalidade da área cultivada. Observações ao microscópio óptico de fragmentos de tecido do sistema vascular das hastes de plantas doentes evidenciaram intenso fluxo bacteriano, confirmando tratar-se de doença bacteriana. Isolamentos realizados em meio nutriente ágar produziram colônias de coloração amarelada, brilhantes, convexas, lisas. A bactéria agente causal era Gram-negativa e produtora de pigmento marrom escuro em meio de cultura. Testes bioquímicos, culturais e fisiológicos confirmaram sua identidade como Xanthomonas fuscans subsp. fuscans (sin. Xanthomonas campestris pv. phaseoli "var. fuscans"). A patogenicidade dos isolados foi confirmada por inoculações artificiais em mudas de feijão cv. Carioca e os reisolamentos efetuados resultaram em colônias semelhantes às originais. Embora descrita na literatura, a infecção sistêmica não é usualmente observada nos plantios de feijoeiro em nosso país. Linhagens bacterianas encontram-se depositadas na Coleção de Culturas do Instituto Biológico (IBSBF) sob os números 2813 e 3028.(AU)

Absence of detection of Xanthomonas axonopodis Pv. Malvacearum in cotton seeds used in the northeast region of Mato Grosso do Sul in a three-based crop season study / Ausência de detecção de Xanthomonas axonopodis Pv. Malvacearum em sementes de algodoeiro usadas na região nordeste de Mato Grosso Do Sul, em um estudo de três safras agrícolas

The aim of this work was to evaluate the presence of the causal agent of angular leaf spot, Xanthomonas axonopodis pv. malvacearum, in 38 cotton seeds samples with or without linter used in the northeast region of Mato Grosso do Sul, Brazil, during the crop seasons of 2008/09, 2009/10 e 2010/11. It was used the semi selective culture media MSSXAN (beef extract - 3 g, peptone - 5 g, soluble starch - 10 g, sucrose - 5g; Tween 80 - 10 mL; CaCl2 - 0.25 g; crystal violet solution at 1% - 150 mL; cephalexin - 50 mg; chlorothalonil - 10 mg; methyl thyophanate - 10 mg; agar ­ 15 g; sterile distilled water - 1000 ml) for the isolation of the bacteria and the suspect colonies of being X. axonopodis pv. malvacearum were purified and submitted to the Gram stain, potassium hydroxide and pathogenicity tests in the leaves of cotton plants of the susceptible genotype NC 53345. The presence of X. a. pv. malvacearum was not detected in the evaluated seed samples.

O objetivo deste trabalho foi avaliar a presença do agente causal da mancha-angular, Xanthomonas axonopodis pv. malvacearum, em 38 amostras de sementes de algodoeiro com ou sem línter utilizadas na região nordeste de Mato Grosso do Sul nas safras 2008/09, 2009/10 e 2010/11. Foi empregado o meio de cultura semi-seletivo MSSXAN (extrato de carne - 3 g; peptona - 5 g; amido solúvel - 10 g; sacarose - 5g; Tween 80 - 10 mL; CaCl2 - 0,25 g; solução de cristal violeta a 1 % - 150 µL; cefalexina - 50 mg; clorothalonil - 10 mg; tiofanato metílico - 10 mg; ágar - 15 g; água destilada esterilizada - 1.000 mL) para o isolamento da bactéria nas sementes dos diferentes lotes e colônias suspeitas de serem X. axonopodis pv. malvacearum foram purificadas e posteriormente submetidas à coloração diferencial de Gram, teste de KOH e patogenicidade em folhas de plântulas suscetíveis de algodoeiro, genótipo NC 53345. Não foi constatada a presença de X. a. pv. malvacearum nas amostras de sementes analisadas.

Genótipos de maracujazeiro-azedo com resistência à bacteriose / Genotypes of passionfruit to resistance to the bacteriose

Este trabalho teve como objetivo avaliar e selecionar genótipos de maracujazeiro-azedo com resistência à bactéria Xanthomonas axonopodis pv. passiflorae. Para a inoculação da bactéria, nas folhas das plantas, foi empregado o método da agulha utilizando-se o delineamento de blocos casualizados, com 4 repetições e 12 plantas por parcela, em esquema de parcela subdividida, com quatro épocas de avaliação na parcela e 18 genótipos na subparcela. Foi avaliada a incidência e a severidade da bacteriose, com base em escala de notas variando de 0 a 4, onde foram classificadas como Resistentes, Medianamente Resistentes, Medianamente Suscetíveis, Suscetíveis e Altamente Suscetíveis. Os genótipos apresentaram variabilidade com relação à resistência, sendo que os genótipos Maracujá Moranga, RC-0-3, Vermelhinho e PES-7 foram selecionados, pois apresentaram mais de 30% de plantas medianamente resistentes na última avaliação. O genótipo MSCA foi considerado resistente com base na severidade. As plantas individuais dos diferentes genótipos que permaneceram resistentes até a última avaliação foram selecionadas e deverão ser novamente avaliadas para a confirmação da resistência à bactéria Xanthomonas axonopodis pv. passiflorae, incluindo diferentes isolados desta bactéria.

This work had as objective to evaluate and to select the reaction of passionfruit genotypes to Xanthomonas axonopodis pv. passiflorae. For the inoculation of the bacterium had been used of the needle. Twelve plants of each of 18 genotypes were evaluated in a randomized block experiment with 4 replicates and four different times of evaluation. It was evaluated the incidence and the severity of bacteriose, on the basis of a note scale of 0 the 4 which were classified as Resistant, Medium Resistant, Medium Susceptible, Susceptible and Highly Susceptible. The genotypes had presented variability with regard to the resistance, being that the genotypes Maracujá Moranga, RC-0-3, Vermelhinho and PES-7 had been selected, since they had more than presented 30% of medium resistant plants in the last evaluation. Only one genotype was considered resistant, on the basis of severity. The individual plants of the different genotypes that had remained resistant until the last evaluation will have to be selected and evaluated for the confirmation of the Xanthomonas axonopodis pv. passiflorae.

In planta detection of Xanthomonas axonopodis pv. commiphorae using fyuA and rpoD genes.

Guggal is tapped for extraction of medicinally important oleo–gum–resin (guggul) by inoculating the stem bark with natural gum suspension containing pathogenic bacterium Xanthomonas axonopodis pv. commiphorae (Xac). The tree dies in the process. In absence of any specific medium for isolation of Xac, it is difficult to assess spread of the pathogen within the plant. A PCR based molecular detection technique using fyuA and rpoD gene specific primers is described here. The primers amplified products only from Xac and not from host tissues or common saprophytes. The method was sensitive enough to produce positive signals for up to 4.4 bacterial cells or 2 pg target DNA per reaction mixture. However, PCR inhibitors present in plant tissues drastically reduced the limit of detection. A simple overnight incubation of surface sterilised plant tissues in nutrient medium was introduced to increase pathogen titre and to overcome this problem. This technique was successfully used to measure spread of Xac in plant tissues away from the site of inoculation. The pathogen showed preference for acropetal movement and did not spread to 7–8 cm below the site of inoculation till 15 days after inoculation. This suggests possibility to manage the disease through plant surgery.

Avaliaçâo da transmissão de Xanthomonas axonopodis pv. malvacearum via semente-algodoeiro em condições de campo, no cerrado sul-mato-grossense / Evaluation of Xanthomonas axonopodis pv. malvacearum transmission from seeds to cotton plants in field conditions, in the cerrado of Mato Grosso do Sul, Brazil

Avaliou-se a transmissão de Xanthomonas axonopodis pv. malvacearum (XAM) via sementeplântula de algodoeiro em condições de campo, no Cerrado sul-mato-grossense,por duas safras agrícolas (2009/10 e 2010/11). Foi empregado o método de inoculação de sementes de algodoeiro, após a validação em condições de laboratório, que consistiu na imersão das sementes em suspensão bacteriana, a 108 ufc.mL-1, por três horas. Foram obtidos lotes com 1, 2, 5, 10 e 20 % de sementes infetadas, que foram semeadas e conduzidas até as avaliações. Após 15, 30 e 45 dias da semeadura, avaliou-se a incidência de plantas com sintomas típicos da mancha-angular. Não foram observadas plantas doentes, o que indicou ausência de transmissão de XAM, das sementes para as plantas emergidas, na região em que este trabalho foi desenvolvido.

AFLP analysis of Xanthomonas axonopodis and X arboricola strains used in xanthan production studies reveal high levels of polymorphism

Amplified fragment length polymorphism (AFLP) was used to analyze the genetic diversity of 14 strains of Xanthomonas arboricola pv. pruni and seven strains of X. axonopodis pv. phaseoli, which are used in xanthan production studies. Relationships identified by the AFLP profiles were assessed for xanthan production capacity, geographical location and host plant. Strains were isolated from 10 different geographic regions in South and Southeast States in Brazil. Data were analyzed for genetic similarity using the Dice coefficient and subjected to UPGMA cluster analysis. A total of 128 AFLP fragments were generated from four primer combinations: EcoRI+C/MseI+0, EcoRI+A/MseI+0, EcoRI+G/MseI+T and EcoRI+G/MseI+A. Of these, 96.1 percent were polymorphic. X. axonopodis pv. phaseoli (S D = 0.27) was shown to be more polymorphic than X. arboricola pv. pruni (S D = 0.58). All 14 pathovar pruni strains were included in a single main group (S D = 0.58), while the pathovar phaseoli strains were divided into three separate groups, with one group containing five strains (S D = 0.38) and two isolated groups (S D = 0.31 and 0.27) composed of only one strain each. Species were distinguished by three and eight specific AFLP markers present in the pathovar phaseoli and the pathovar pruni, respectively. For the unique strain without xanthan production capacity (X. axonopodis pv. phaseoli str. 48), nine specific AFLP bands were found. There was no evidence that geographic area or host plant influenced genetic heterogeneity. Correlations between AFLP patterns and xanthan production capacity were found in some strains, but were not consistent enough to establish a relationship.

Controle de crestamento bacteriano comum (Xanthomonas axonopodis Ppv. phaseoli) e alterações bioquímicas em feijoeiro induzidas por Pycnoporus sanguineus / Control of bacterial blight (Xanthomonas axonopodis pv. phaseoli) and biochemical analyses of bean resistance treated with Pycnoporus sanguineus extracts

O objetivo deste trabalho foi verificar as atividades antibacteriana e indutora de resistência de extratos de Pycnoporus sanguineus para controle do crestamento bacteriano comum, causado por Xanthomonas axonopodis pv. phaseoli, em feijoeiro. In vitro foram utilizados extratos aquosos de basidiocarpo, micélio e filtrado de cultura de P. sanguineus nas concentrações de 1, 5, 10, 15 e 20%, além das testemunhas água, acibenzolar-S-metil (ASM - 125 mg i.a. L-1) e antibiótico (22,5 mg L-1 de oxitetraciclina + 225 mg L-1 de estreptomicina). In vivo foram realizadas avaliações de severidade e atividade de peroxidase, polifenoloxidase, ß-1,3 glucanase e fenilalanina amônia-liase, com o uso de extrato aquoso de micélio e de basidiocarpo e filtrado de cultura de P. sanguineus a 5% e 10%. Verificou-se atividade antibacteriana apenas para o filtrado de cultura em concentrações acima de 15% e para o extrato de basidiocarpo nas concentrações de 1 a 20%. In vivo, os resultados indicaram o potencial de extratos de basidiocarpos de P. sanguineus para o controle de X. axonopodis pv. phaseoli em feijoeiro, com redução média de 56% na severidade, o que pode ter ocorrido tanto por atividade antimicrobiana direta quanto por indução de resistência, envolvendo principalmente a ativação das enzimas de defesa vegetal peroxidase e polifenoloxidase.

The aim of this work was to verify the antimicrobial and resistance induction activities of Pycnoporus sanguineus extracts for the control of common bacterial blight caused by Xanthomonas axonopodis pv. phaseoli. In vitro assays were performed using aqueous extracts from basidiocarp, mycelium and culture filtrate of P. sanguineus in concentrations of 1, 5, 10, 15 and 20%, with water, acibenzolar-S-methyl (ASM - 125 mg a.i. L-1) and antibiotic (oxytetracycline 22.5 mg L-1 + streptomycin 225 mg L-1) as control treatments. For the in vivo assays the disease severity and the activities of peroxidase, polyphenol oxidase, ß-1,3 glucanase and phenylalanine ammonia-lyase were evaluated using extracts of mycelium, basidiocarp and culture filtrate of P. sanguineus at 5% and 10%. Antibacterial activity was verified only for culture filtrate in concentrations above 15% and for concentrations of 1% to 20% of basidiocarp extract. The results of the in vivo assays indicated the potential of basidiocarps extracts from P. sanguineus for the control of X. axonopodis pv. phaseoli in beans, with an average severity reduction of 56%, which may have been due either to direct antimicrobial activity or to resistance induction involving mainly the activation of the plant defense enzymes peroxidase and polyphenol oxidase.

Estudos estruturais e de interações proteína-proteína envolvendo componentes de um sistema de secreção do tipo IV de Xanthomonas axonopodis pv. citri / Structural and protein-protein interaction studies of type IV secretion system components from Xanthomonas axonopodis pv. citri

Xanthomonas axonopodis pv. citri (Xac) é o causador do cancro de plantas cítricas. Entre os potenciais fatores de virulência codificados por Xac, está o Sistema de Secreção do Tipo IV (T4SS), um grande complexo multiprotéico que atravessa o periplasma e as membranas interna e externa de bactérias Gram-negativas. O T4SS está envolvido com secreção de proteínas e/ou DNA para o meio extracelular ou diretamente no interior da célula do hospedeiro. Este Sistema requer tipicamente 12 proteínas para realizar suas funções: VirB1-VirB11 e VirD4. O T4SS codificado pelo cromossomo de Xac está aparentemente incompleto, devido a não codificar nenhuma proteína com similaridade de seqüência a VirB7. Os objetivos deste trabalho são estudar a estrutura, função e interações das proteínas do T4SS de Xanthomonas. Foram clonados 23 genes que codificam proteínas ou domínios relacionados ao T4SS, e os polipeptídeos foram produzidos de forma recombinante em E. coli. Treze deles foram purificados e submetidos a estudos estruturais, espectroscópicos e de interações proteína-proteína. A estrutura em solução de Xac262224-139 foi resolvida, apresentando uma região N-terminal desenovelada de aproximadamente 30 resíduos e um domínio globular. Este polipeptídeo oligomeriza em troca química rápida na escala de tempo de RMN e o seu N-terminal desenovelado reconhece o domínio C-terminal de VirB9 (VirB9154-255) em troca lenta. Análise de RMN demonstrou que VirB9154-255 possui uma estrutura flexível em solução, sofrendo uma marcante mudança conformacional na presença de Xac262224-139. Ambas proteínas se tornam rígidas após a interação. Xac2622 é o equivalente a VirB7 em Xanthomonas, baseado na localização do seu gene no lócus do T4SS, localização subcelular predita do polipeptídeo codificado e sua interação com VirB9...

Xanthomonas axonopodis pv. citri (Xac) is a gram-negative bacterial phytopathogen that infects citrus. One possible virulence determinant is a chromosomally encoded Type IV Secretion System (T4SS), a multiprotein complex that spans the bacterial periplasm and both inner and outer membranes. The T4SS is used by some bacteria to secrete proteins and/or DNA to the extracellular milieu or the host interior. The model T4SS from Agrobacterium tumefaciens is made up of twelve structural proteins: VirB1-VirB11 and VirD4. The Xanthomonas T4SS is apparently incomplete because of the lack of a polypeptide with sequence similarity to VirB7. The aim of this project is the study of structure-function relationships in the Xanthomonas T4SS. Twenty-three T4SS protein-coding genes, including full-length proteins or domains, were cloned and the proteins were produced in different E. coli strains. Thirteen polypeptides were purified and some of them were submitted to structural, spectroscopic and protein-protein interaction studies. We used NMR to solve the solution structure of Xac262224-139 which consists of an unfolded N-terminal segment of ~30 residues followed by a globular domain. Xac262224-139 oligomerizes in fast exchange at the NMR time scale and interacts via its unfolded N-terminus with the VirB9 C-terminus (VirB9154-255) in slow exchange. NMR analysis showed that VirB9154-255 has a flexible structure in solution. However, this polypeptide undergoes a significant conformational modification in the presence of Xac2622,24-139 and both proteins become rigid upon interaction. Xac2622 is the Xanthomonas VirB7, based on the chromosomal localization of its gene, predicted subcellular localization and protein interaction analysis. But surprisingly, unlike other VirB7 proteins, Xac2622 has an extra C-terminal folded domain whose topology and structure are strikingly similar to that of periplasmic domains found in outer membrane proteins of many bacterial Secretion Systems...

Defense mechanisms of Solanum tuberosum L. in response to attack by plant-pathogenic bacteria

The natural resistance of plants to disease is based not only on preformed mechanisms, but also on induced mechanisms. The defense mechanisms present in resistant plants may also be found in susceptible ones. This study attempted to analyze the metabolic alterations in plants of the potato Solanum tuberosum L. cv. Agata that were inoculated with the incompatible plant-pathogenic bacteria X. axonopodis and R. solanacearum, and the compatible bacterium E. carotovora. Levels of total phenolic compounds, including the flavonoid group, and the activities of polyphenol oxidase (PPO) and peroxidase (POX) were evaluated. Bacteria compatibility was evaluated by means of infiltration of tubers. The defense response was evaluated in the leaves of the potato plants. Leaves were inoculated depending on their number and location on the stem. Multiple-leaf inoculation was carried out on basal, intermediate, and apical leaves, and single inoculations on intermediate leaves. Leaves inoculated with X. axonopodis and with R. solanacearum showed hypersensitive responses within 24 hours post-inoculation, whereas leaves inoculated with E. carotovora showed disease symptoms. Therefore, the R. solanacearum isolate used in the experiments did not exhibit virulence to this potato cultivar. Regardless of the bacterial treatments, the basal leaves showed higher PPO and POX activities and lower levels of total phenolic compounds and flavonoids, compared to the apical leaves. However, basal and intermediate leaves inoculated with R. solanacearum and X. axonopodis showed increases in total phenolic compounds and flavonoid levels. In general, multiple-leaf inoculation showed the highest levels of total phenolics and flavonoids, whereas the single inoculations resulted in the highest increase in PPO activity. The POX activity showed no significant difference between single- and multiple-leaf inoculations. Plants inoculated with E. carotovora showed no significant increase ...

Production of the refolded oligopeptide-binding protein (OppA) encoded by the citrus pathogen Xanthomonas axonopodis pv. citri

The oligopeptide-binding protein, OppA, binds and ushers oligopeptide substrates to the membrane-associated oligopeptide permease (Opp), a multi-component ABC-type transporter involved in the uptake of oligopeptides expressed by several bacterial species. In the present study, we report the cloning, purification, refolding and conformational analysis of a recombinant OppA protein derived from Xanthomonas axonopodis pv. citri (X. citri), the etiological agent of citrus canker. The oppA gene was expressed in Escherichia coli BL21 (DE3) strain under optimized inducing conditions and the recombinant protein remained largely insoluble. Solubilization was achieved following refolding of the denatured protein. Circular dichroism analysis indicated that the recombinant OppA protein preserved conformational features of orthologs expressed by other bacterial species. The refolded recombinant OppA represents a useful tool for structural and functional analyses of the X. citri protein.

In silico identification of potential chaperone genes that belong to type III and type IV secretion systems in Xanthomonas axonopodis pv citri

The secretion of bacterial virulence factors and flagellar components requires the assistance of specific type III and flagellar chaperones. Standard computational annotation of the genome of Xanthomonas axonopodis pv citri, a plant pathogen that causes citrus canker, initially did not identify any genes belonging to these chaperone categories since the primary sequence homology between them was very low. However, in a search for hypothetical proteins with characteristics similar to these chaperones, we have now identified 30 chromosomal and 10 plasmidial potential genes encoding chaperones belonging to types III/IV, and flagellar secretion systems in this organism. The significance of these findings is discussed.