Development and application of in vivo expression technology (IVET) for analysing microbial gene expression in complex environments

Establishing the mechanisms by which microbes interact with their environment, including eukaryotic hosts, is a major challenge that is essential for the economic utilisation of microbes and their products. Techniques for determining global gene expression profiles of microbes, such as microarray analyses, are often hampered by methodological restraints, particularly the recovery of bacterial transcripts (RNA) from complex mixtures and rapid degradation of RNA. A pioneering technology that avoids this problem is In Vivo Expression Technology (IVET). IVET is a 'promoter-trapping' methodology that can be used to capture nearly all bacterial promoters (genes) upregulated during a microbe-environment interaction. IVET is especially useful because there is virtually no limit to the type of environment used (examples to date include soil, oomycete, a host plant or animal) to select for active microbial promoters. Furthermore, IVET provides a powerful method to identify genes that are often overlooked during genomic annotation, and has proven to be a flexible technology that can provide even more information than identification of gene expression profiles. A derivative of IVET, termed resolvase-IVET (RIVET), can be used to provide spatio-temporal information about environment-specific gene expression. More recently, niche-specific genes captured during an IVET screen have been exploited to identify the regulatory mechanisms controlling their expression. Overall, IVET and its various spin-offs have proven to be a valuable and robust set of tools for analysing microbial gene expression in complex environments and providing new targets for biotechnological development.

Testing temperature-induced proteomic changes in the plant-associated bacterium Pseudomonas fluorescens SBW25.

Traits used by bacteria to enhance ecological performance in natural environments are not well understood. Recognizing that the saprophytic plant-colonizing bacterium Pseudomonas fluorescens SBW25 experiences temperatures in its natural environment significantly cooler than the 28°C routinely used in the laboratory, we identified proteins differentially expressed between 28°C and the more environmentally relevant temperature of 14°C. Of 2102 protein isoforms, 32 were temperature responsive and identified by mass spectrometry. Seven of these (OmpR, MucD, GuaD, OsmY and three of unknown function, Tee1, Tee2 and Tee3) were selected for genetic and ecological analyses. In each instance, changes in protein expression with temperature were mirrored by parallel transcriptional changes. The fitness contribution of the genes encoding each of the seven proteins was larger at 14°C than 28°C and included two cases of trade-offs (enhanced fitness at one temperature and reduced fitness at the other – mucD and tee2 deletions). The relationship between the fitness effects of genes in vitro and in vivo was variable, but two temperature-responsive genes – osmY and mucD – contribute substantially to the ability of P. fluorescens to colonize the plant environment.

The biosurfactant viscosin produced by Pseudomonas fluorescens SBW25 aids spreading motility and plant growth promotion

Food security depends on enhancing production and reducing loss to pests and pathogens. A promising alternative to agrochemicals is the use of plant growth-promoting rhizobacteria (PGPR), which are commonly associated with many, if not all, plant species. However, exploiting the benefits of PGPRs requires knowledge of bacterial function and an in-depth understanding of plant-bacteria associations. Motility is important for colonization efficiency and microbial fitness in the plant environment, but the mechanisms employed by bacteria on and around plants are not well understood. We describe and investigate an atypical mode of motility in Pseudomonas fluorescens SBW25 that was revealed only after flagellum production was eliminated by deletion of the master regulator fleQ. Our results suggest that this ‘spidery spreading’ is a type of surface motility. Transposon mutagenesis of SBW25ΔfleQ (SBW25Q) produced mutants, defective in viscosin production, and surface spreading was also abolished. Genetic analysis indicated growth-dependency, production of viscosin, and several potential regulatory and secretory systems involved in the spidery spreading phenotype. Moreover, viscosin both increases efficiency of surface spreading over the plant root and protects germinating seedlings in soil infected with the plant pathogen Pythium. Thus, viscosin could be a useful target for biotechnological development of plant growth promotion agents.

Transcriptomic analysis of Enterohaemorrhagic Escherichia coli O157:H7 in response to plant extracts

Enterohaemorrhagic Escherichia coli (EHEC) are a group of food and contact-borne pathogens responsible for haemorrhagic colitis. The bacteria can be transmitted by contaminated meat, but importantly, also by plants. The bacteria can use plants as an alternative host, where they associate with both the leaves and the roots. Colonisation in the rhizosphere of plants is thought to be the main habitat for colonisation. Four different plant species, commonly associated with EHEC outbreaks, were infected with EHEC O157:H7 isolates Sakai and TUV 93-0 over ten days to assess the colonisation potential of the bacteria in both the phyllosphere and rhizosphere of plants. The rhizosphere was found to sustain a higher population level of bacteria over time in comparison to the phyllosphere, yet both strains were unable to utilize root exudates for growth. Global gene expression changes of EHEC O157:H7 strain Sakai were measured in response to plant extracts such as leaf lysates, root exudates and leaf cell wall polysaccharides from spinach cultivar Amazon and lettuce cultivar Salinas. Microarrays analysis showed a significant change in expression of 17 % of genes on exposure to leaf lysates of spinach. A more specific response was seen to spinach leaf cell wall polysaccharides with only a 1.5 % change. In contrast, when exposed to lettuce leaf cell wall polysaccharides a higher change of 4.8 % was seen. Genes that were differentially expressed belonged to multiple functional groups, including metabolism, indicating the utilization of plant-specific polysaccharides. Several areas of further investigation have been determined from this project, including the importance of culturing bacterial strains at a relevant temperature, the proposed lack of the type III secretion system in plant colonization by EHEC O157:H7 and the utilization of plant components for growth and persistence in the plant environment.

Infestation of froghopper nymphs changes the amounts of total phenolics in sugarcane

The increased rate of sugarcane harvest without previous burn has provided a very favorable environment to the froghopper Mahanarva fimbriolota (Stal, 1854), with high Moisture and low temperature variation. Few works have Studied the response of sugarcane to this pest, so little is known about resistant cultivars. Plant phenolics are widely studied compounds because of their known antifierbivore effect. This research aims to determine if the attack of M.fimbriolata nymphs stimulates the acccumulation of total phenolics in sugarcane. The experiment was carried Out in greenhouse and arranged in completely randomized design, in a 3 x 2 x 4 factorial with three replications. Second instar nymphs of M. fimbriolota were infested at the following rates: control, 2-4 and 4-8 nymphs per pot (first-second infestations, respectively). Pots were covered with nylon net and monitored daily to isolate the effect of leaf Sucking adults. Leaf and root samples were collected and kept frozen in liquid nitrogen until analyses. Infested plants showed higher levels of phenolics in both root and leaf tissues. In roots, the cultivar SP80-1816 accumulated more phenolic compounds in response to the infestation of M. fimbriolata. on the other hand, higher levels were found in leaves and roots of control plants of SP86-42, which might be an indication of a non-preference mechanism. The increase of total phenolics in sugarcane infested with root-sucking froghopper nymphs does not seem to be useful to detect the resistance to this pest.

Adaptabilidade e estabilidade fenotípica de cultivares de cenoura

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Seleção de progênies de meios-irmãos do composto Isanão VF-1 de milho na safra e safrinha

O aumento significativo da produção de milho na segunda safra no Brasil, principalmente no centro-sul do país, têm estimulado os programas de melhoramento da cultura a selecionar genótipos que sejam adaptáveis às condições climáticas das diferentes épocas de semeadura. Nesse sentido, o objetivo do presente trabalho foi quantificar a interação progênies x épocas de semeadura e verificar seus reflexos no progresso genético com o uso de índice de seleção multivariado para seleção de progênies do Composto Isanão VF-1 de milho. As semeaduras foram realizadas na segunda safra em 2004 e na primeira safra do ano agrícola 2004/05. Foram utilizadas 71 progênies de meios irmãos avaliadas em blocos ao acaso, com três repetições. Os caracteres avaliados foram: altura de plantas, altura de espigas, tombamento, prolificidade e rendimento de grãos. Realizaram-se a decomposição da interação progênies x épocas e foram estimados os ganhos pelo índice de seleção descrito por Mulamba e Mock. Houve predomínio da interação do tipo simples para maioria dos caracteres, exceto para prolificidade, que revelou 86% de interação do tipo complexa. Pelo índice de Mulamba e Mock, os ganhos proporcionais mais adequados para o conjunto de caracteres avaliados foi obtido pelos pesos econômicos atribuídos por tentativas. Os ganhos preditos foram de 1,41, 0,86, -13,03, 9,54 e 16,12% para altura de planta, altura de espiga, tombamento, prolificidade e rendimento de grãos, respectivamente.

Genetic group x ambient temperature interaction effects on physiological responses and growth performance of rabbits

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Serpentine leafminer (Liriomyza trifolii) on potato (Solanum tuberosum): field observations and plant photosynthetic responses to injury

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Testes multiambientais na seleção de genótipos de arroz utilizando o modelo de regressão nos sítios ou locais

A análise da interação genótipo x ambiente utilizda no melhoramento de plantas tem sofrido mudanças na última década, melhorando a sua eficiência quanto à seleção dos genótipos sob diferentes condições ambientais. O objetivo deste trabalho foi analisar a produtividade e estabilidade de 12 genótipos de arroz em oito ambientes, durante os anos 2005 e 2006, na Colômbia. O delineamento utilizado foi o de blocos ao acaso com quatro repetições. Os parâmetros de estabilidade fenotípica e o agrupamento dos ambientes foram estimados pelo estudo da interação genótipo x ambiente, segundo o método SREG (Regressão nos sítios ou locais) e seu gráfico biplot (GGE). As análises estatísticas indicaram diferenças significativas (com 5% de probabilidade de erro) entre genótipos e entre ambientes e significância (com 5% de probabilidade de erro) da interação genótipo x ambiente, sugerindo uma resposta diferente dos genótipos nos vários ambientes. No método SREG, os dois primeiros componentes principais da interação explicaram 75,29% da interação. Os genótipos 400094, 350361 e a variedade Fedearroz 50 foram considerados os de maior produtividade. Segundo o gráfico biplot GGE, os ambientes La Libertad e Escobal foram os mais favoráveis para o cultivo do arroz.

A Bayesian approach for assessing the stability of genotypes

Several statistical models can be used for assessing genotype X environment interaction (GEI) and studying genotypic stability. The objectives of this research were to show how (i) to use Bayesian methodology for computing Shukla's phenotypic stability variance and (ii) to incorporate prior information on the parameters for better estimation. Potato [Solanum tuberosum subsp. andigenum (Juz. & Bukasov) Hawkes], wheat (Triticum aestivum L.), and maize (Zea mays L.) multi environment trials (MET) were used for illustrating the application of the Bayes paradigm. The potato trial included 15 genotypes, but prior information for just three genotypes was used. The wheat trial used prior information on all 10 genotypes included in the trial, whereas for the maize trial, noninformative priors for the nine genotypes was used. Concerning the posterior distribution of the genotypic means, the maize MET with 20 sites gave less disperse posterior distributions of the genotypic means than did the posterior distribution of the genotypic means of the other METs, which included fewer environments. The Bayesian approach allows use of other statistical strategies such as the normal truncated distribution (used in this study). When analyzing grain yield, a lower bound of zero and an upper bound set by the researcher's experience can be used. The Bayesian paradigm offers plant breeders the possibility of computing the probability of a genotype being the best performer. The results of this study show that although some genotypes may have a very low probability of being the best in all sites, they have a relatively good chance of being among the five highest yielding genotypes.

Polymorphisms of DNA repair genes XRCC1 and XRCC3, interaction with environmental exposure and risk of chronic gastritis and gastric cancer

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Nicotiana tabacum as an experimental host for the study of plant-Xylella fastidiosa interactions

Xylella fastidiosa causes citrus variegated chlorosis (CVC). Information generated from the X. fastidiosa genome project is being used to study the underlying mechanisms responsible for pathogenicity. However, the lack of an experimental host other than citrus to study plant-X. fastidiosa interaction has been an obstacle to accelerated progress in this area. We present here results of three experiments that demonstrated that tobacco could be an important experimental host for X. fastidiosa. All tobacco plants inoculated with a citrus strain of X. fastidiosa expressed unequivocal symptoms, consisting of orange leaf lesions, approximately 2 months after injection of the pathogen. CVC symptoms were observed in citrus 3 to 6 months after inoculation. The pathogen was readily detected in symptomatic tobacco plants by polymerase chain reaction (PCR) and phase contrast microscopy. In addition, X. fastidiosa was reisolated on agar plates in 4 of 10 plants. Scanning electron microscopy analysis of cross sections of stems and petioles revealed the presence of rod shaped bacteria restricted to the xylem of inoculated plants. The cell size was within the limit typical of X. fastidiosa.

Size and lipid content of nonmyrmecochorous diaspores: Effects on the interaction with litter-foraging ants in the Atlantic rain forest of Brazil

Ants are often attracted to diaspores not adapted for dispersal by ants. These diaspores may occasionally benefit from this interaction. We selected six nonmyrmecochorous plant species (Virola oleifera, Eugenia stictosepala, Cabralea canjerana, Citharexylum myrianthum, Alchornea glandulosa and Hyeronima alchorneoides) whose diaspores differ in size and lipid content, and investigated how these features affect the outcome of ant-diaspore interactions on the floor of a lowland Atlantic forest of Southeast Brazil. A total of 23 ant species were seen interacting with diaspores on the forest floor. Ants were generally rapid at discovering and cleaning the diaspore pulp or aril. Recruitment rate and ant attendance were higher for lipid-rich diaspores than for lipid-poor ones. Removal rate and displacement distance were higher for small diaspores. The large ponerine ant Pachycondyla striata, one of the most frequent attendants to lipid-rich arillate diaspores, transported the latter into their nests and discarded clean intact seeds on refuse piles outside the nest. Germination tests with cleaned and uncleaned diaspores revealed that the removal of pulp or aril may increase germination success in Virola oleifera, Cabralea canjerana, Citharexylum myrianthum and Alchornea glandulosa. Gas chromatography analyses revealed a close similarity in the fatty acid composition of the arils of the lipid-rich diaspores and the elaiosome of a typical myrmecochorous seed (Ricinus communis), corroborating the suggestion that some arils and elaiosomes are chemically similar. Although ant-derived benefits to diaspores - secondary dispersal and/or increased germination - varied among the six plant species studied, the results enhanced the role of ant-diaspore interactions in the post-dispersal fates of nonmyrmecochorous seeds in tropical forests. The size and the lipid-content of the diaspores were shown to be major determinants of the outcome of such interactions.

Polymorphisms of DNA repair genes XRCC1 and XRCC3, interaction with environmental exposure and risk of chronic gastritis and grastic cancer

Aim: To evaluate the association between polymorphisms XRCC1 Arg194Trp and Arg399Gln and XRCC3 Thr241Met and the risk for chronic gastritis and gastric cancer, in a Southeastern Brazilian population. Methods: Genotyping by PCR-RFLP was carried out on 202 patients with chronic gastritis (CG) and 160 patients with gastric cancer (GC), matched to 202 (C1) and 150 (C2) controls, respectively. Results: No differences were observed among the studied groups with regard to the genotype distribution of XRCC1 codons 194 and 399 and of XRCC3 codon 241. However, the combined analyses of the three variant alleles (194Trp, 399Gln and 241Met) showed an increased risk for chronic gastritis when compared to the GC group. Moreover, an interaction between the polymorphic alleles and demographic and environmental factors was observed in the CG and GC groups. XRCC1 194Trp was associated with smoking in the CG group, while the variant alleles XRCC1 399Gln and XRCC3 241Met were related with gender, smoking, drinking and H pylori infection in the CG and GC groups. Conclusion: Our results showed no evidence of a rela-tionship between the polymorphisms XRCC1 Arg194Trp and Arg399Gln and XRCC3 Thr241Met and the risk of chronic gastritis and gastric cancer in the Brazilian population, but the combined effect of these variants may interact to increase the risk for chronic gastritis, considered a premalignant lesion. Our data also indicate a gene-environment interaction in the susceptibility to chronic gastritis and gastric cancer. © 2005 The WJG Press and Elsevier Inc. All rights reserved.