Mean winter air temperatures have risen by 2.5˚C over the last 50 years in the northeastern U.S., reducing mean annual winter snowpack depth by 26 cm and the duration of winter snow cover by four days per decade. Because snow cover insulates soil from below-freezing air temperatures, continued declines in snowpack depth are projected to be accompanied by colder winter soil temperatures and more frequent soil freeze-thaw events. Soil bacteria and fungi will play a significant role in the forest ecosystem response to snowpack loss because they are the primary agents that carry out soil organic matter decomposition and soil nutrient cycling. Additionally, the effect of winter snowpack decline on soil bacterial and fungal communities may act indirectly via winter climate change effects on plant roots. The objectives of my dissertation research were to first determine the effect that reductions in winter snow cover has on microbial exoenzyme activity, microbial respiration, net nitrogen (N) mineralization, and net nitrification rates in two mixed-hardwood forests (Harvard Forest, MA and Hubbard Brook Experimental Forest, NH). Additionally, I sought to determine the relative role that abiotic factors (i.e., winter snow cover or soil frost) versus biotic factors (i.e., altered root-microbe interactions) contribute to overall changes in soil biogeochemical processes as winter snow cover declines. I found that winter snow depth and duration are related positively to microbial exoenzyme activity and microbial respiration following snowmelt in spring, but this relationship is transient and attenuates into the growing season. By contrast, soil freeze-thaw events during winter result in persistent declines in microbial oxidative enzyme activity that are not compensated for by warming soils during the growing season. Together, these results suggest that loss of winter snow cover will result in lower rates of nutrient cycling in northeastern U.S. hardwood forests. Tree roots interact with winter snow depth to affect net mineralization and nitrification rates, as well as bacterial and fungal community composition. Thus, winter climate change portends a reorganization of root-microbe interactions with important consequences for soil biogeochemical cycling in mixed hardwood forests of the northeastern U.S.

The relationship between a pathogen and its host is a complex series of events that occurs at the molecular level and is controlled by transcriptional and protein interactions. To facilitate the understanding of these mechanisms in Aspergillus flavus and Zea mays, three approaches were taken: 1) the development of a predicted interactome for Z. mays (PiZeaM), 2) the development of co-expression networks for Z. mays and A. flavus from RNA-seq data, and 3) the development of causal inference networks depicting interactions between the host and the pathogen. PiZeaM is the genome-wide roadmap of protein-protein interactions that occur within Z. mays. PiZeaM helps create a novel map of the interactions in Z. mays in response to biotic and abiotic stresses. To further support the predicted interactions, an analysis of microarray-based gene expression was used to produce a gene co-expression network. PiZeaM was able to capture conserved resistance pathways involved involved in the response to pathogens, abiotic stress and development. Gene Co-expression networks were developed by the simultaneous use of correlations to develop networks for differentially expressed genes, resistance marker genes, pathogenicity genes, and genes involved is secondary metabolism in Z. mays and A. flavus. From these networks, correlation and anti-correlation of host and pathogen gene expression was detected, revealing genes that potentially interact at different stages of pathogenesis. Finally, causal gene regulatory relationships were inferred using partial correlation analysis of Z. mays infected with A. flavus over a 3 day period. The gene regulatory network (GRN) sheds light on the specifics of the mechanisms of pathogenesis and resistance that govern the Z. mays-A. flavus interaction. The direct product of this research is the understanding of key transcription factors and signaling genes involved in resistance. This body of research highlights how PPIs and GRNs can be utilized to identify biomarkers and gene functions in both Z. mays and A. flavus.

Strains of the well-studied plant pathogen Pseudomonas syringae show large differences in their ability to colonize plants epiphytically and to inflict damage to hosts. Additionally, P. syringae can infect some sap-sucking insects and at least one P. syringae strain is highly virulent to insects, causing death to most individuals within as few as 4 days and growing to high population densities within insect hosts. The likelihood of agricultural pest insects coming into contact with transient populations of P. syringae while feeding on plants is high, yet the ecological implications of these interactions are currently not well understood as virulence has not been tested across a wide range of strains. To investigate virulence differences across strains we exposed the sweet potato whitefly, Bemisia tabaci, and the pea aphid, Acyrthosiphon pisum, both of which are cosmopolitan agricultural pests, to 12 P. syringae strains. We used oral inoculations with bacteria suspended in artificial diet in order to assay virulence while controlling for other variables such as differences in epiphytic growth ability. Generally, patterns of pathogenicity remain consistent across the two species of hemipteran insects, with bacterial strains from phylogroup II, or genomospecies 1, causing the highest rate of mortality with up to 86% of individuals dead after 72 h post infection. The rate of mortality is highly variable across strains, some significantly different from negative control treatments and others showing no discernable difference. Interestingly, one of the most pathogenic strains to both aphids and whiteflies (Cit7) is thought to be nonpathogenic on plants. We also found Cit7 to establish the highest epiphytic population after 48 h on fava beans. Between the nine P. syringae strains tested for epiphytic ability there is also much variation, but epiphytic ability was positively correlated with pathogenicity to insects, suggesting that the two traits may be linked and that strains likely to be found on plants may often be entomopathogenic. Our study highlights that there may be a use for epiphytic bacteria in the biological control of insect crop pests. It also suggests that interactions with epiphytic bacteria could be evolutionary and ecological drivers for hemipteran insects.

No description available.

Crown gall disease of plants is caused by introduction of foreign DNA into susceptible plant cells by strains of Agrobacterium tumefaciens. The expression of bacterial virulence genes is triggered by chemicals present in plant wound exudates. The exudates contain a number of phenolic compounds which act as chemical signals inducing expression of a number of genes directing the DNA transfer process. These are the virulence or vir genes, and vir::lac reporter gene fusions have been widely used to assay vir gene induction in Agrobacterium tumefaciens strains. Using such strains to monitor vir gene expression, Stachel et al. (1985) isolated from Nicotiana tabacum two active acetophenones: 3,5-dimethoxy-4-hydroxyacetophenone, ("acetosyringone" or AS), and α-hydroxy-3,5-dimethoxy-4-hydroxy-acetophenone, ("hydroxyacetosyringone" or HO-AS). However, in vitro assay results suggested that other more common compounds also exhibited activity (Spencer and Towers, 1988). This analysis of structure-activity relationships of induced vir expression in A. tumefaciens was presented in a previous thesis (Paul Spencer, M.Sc. thesis). The results revealed that a variety of commonly occurring plant phenolic compounds were capable of activating vir genes. In addition to the acetophenones, a variety of benzoic and cinnamic acid derivatives, and even a few chalcones of appropriate ring substitution were active. This thesis reports the isolation and identification of a number of these compounds in plant wound exudates. Some Agrobacterium tumefaciens strains are restricted in host range to certain grapevine cultivars. Subsequent to the development of a convenient and sensitive plate-bioassay method, a strongly active component in grapevine wound exudates was purified. A newly described vir-inducing phenolic compound was isolated from a number of Vitis cultivars using gel filtration, thin layer and high pressure liquid chromatographies. This was identified as syringic acid methyl ester (3,5-dimethoxy-4-hydroxybenzoic acid, methyl ester), using mass spectrometry. However, the presence of this compound in grapevine wound exudates does not provide a simple explanation for host range limitation of grapevine strains since it induces vir gene expression in both limited and wide host range strains of A. tumefaciens. Interestingly, neither AS nor HO-AS were present in grapevine-derived extracts. A convenient polyamide column chromatographic method was subsequently developed to permit rapid purification of plant-derived vir gene inducing mixtures, which were detected using the newly developed plate bioassay. Derivatized polyamide fractions were then analysed by combined gas chromatography-mass spectrometry (GC-MS). GC-MS proved to be an ideal means for the identification of the phenolic components in partially purified extracts. Examination of wound exudates from a range of host and non-host species revealed that the production of the acetophenones is restricted to members of the Solanaceae. Some experiments focussed on the biosynthetic precursors of the acetophenones in Nicotiana species. Wound exudates of the majority of species belonging to other plant families contained benzaldehydes and/or benzoic and cinnamic acid derivatives. The induction of virE gene expression was examined in the related Agrobacterium species, A. rhizogenes. To do this, the virE::lacZ gene fusion plasmid pSM358cd was introduced into A. rhizogenes A4 by triparental mating and the strain "A4/pSM358cd" was used to analyze vir activation. Acetophenones, chalcones, benzaldehydes, and benzoic and cinnamic acid derivatives were found to activate vir genes in A. rhizogenes. / Science, Faculty of / Botany, Department of / Graduate

O comércio mundial de enzimas industriais estava estimado em 2.3 bilhões de dólares entre detergentes (U$ 789 milhões), aplicações alimentícias (U$ 634 milhões), agricultura (U$ 237 milhões), entre outros. Neste contexto, as enzimas lipolíticas estão atraindo enorme atenção devido ao seu potencial biotecnológico, visto que estas podem catalisar múltiplas reações (hidrólise, acidólise, interesterificação e glicerólise). Enzimas lipolíticas de origem microbiana são economicamente atrativas por serem biodegradáveis, atuarem normalmente em condições brandas, e serem quimio-seletivas propiciando à indústria farmacêutica a obtenção de drogas com efeito colateral reduzido. Neste projeto, quatro genes potenciais codificadores de esterases/lipases, advindos de uma biblioteca metagenômica de um consórcio microbiano degradador de óleo diesel, foram clonados em vetores de expressão e expressos em Escherichia coli BL21 (DE3), e as proteínas correspondentes foram submetidas a ensaios funcionais e estruturais. / The global trade of industrial enzymes is estimated at 2.3 billion U.S. dollars, divided mainly between detergents (US$ 789 million), food applications (US$ 634 million), and agriculture (US$ 237 million). Within this trade, lipolytic enzymes have attracted enormous attention because of their biotechnological potential as catalysts of multiple reaction types (including hydrolysis, acidolysis, interesterification and glycerolysis). Lipolytic enzymes of microbial origin are economically attractive because they are easily biodegradable, usually act in mild conditions, and are chemo-selective, providing the pharmaceutical industry a method for obtaining drugs with reduced side effects. In this project, four individual genes encoding putative esterases/lipases identified in a metagenomic library obtained from a microbe consortium isolated from diesel oil-contaminated soil were cloned into expression vectors and expressed in Escherichia coli BL21 (DE3), and their corresponding recombinant proteins were used for functional and structural studies.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13344号 / 論農博第2887号 / 新制||農||1079(附属図書館) / 学位論文||R2||N5251(農学部図書室) / (主査)教授 小川 順, 教授 木岡 紀幸, 教授 栗原 達夫 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

No description available.

Food-borne infections are a major source of mortality and morbidity. Salmonella causes the highest number of Food-borne bacterial infections in the US. This work contributes towards developing strategies to control Salmonella by (a) defining receptors used by Salmonella to adhere to and invade the host epithelium; (b) developing a host receptor based rapid detection method for the pathogen in food matrix; (C) and defining mechanisms of how probiotics can help alleviate Salmonella-induced cell death in the host epithelium. We developed a cell-cell crosslinking method to discover host-microbe receptors, and discovered three new receptor-ligand interactions. Interaction of Salmonella Ef-Tu with Hsp90 from epithelial cells mediated adhesion, while interaction of Salmonella Ef-Tu with two host proteins that negatively regulate membrane ruffling (myosin phosphatase and alpha catenin) mediated adhesion and invasion. We also showed the role of host ganglioside GM1 in mediating invasion of epithelial cells by Salmonella. Further we exploited pathogen affinity for immobilized gangliosides to concentrate them out of solution and from complex food matrices for detection by qPCR. A sensitivity of 4 CFU/ml (3 hours) in samples without competing microflora was achieved. Samples with competing microflora had a sensitivity of 40,000 CFU/ml. Next we screened several probiotic strains for pathogen exclusion potential and found that Bifidobacterium longum subspp. infantis showed the highest potential for Salmonella enterica subspp. enterica ser. Typhimurium exclusion in a caco-2 cell culture model. B. infantis shared its binding specificity to ganglioside GM1 with S. ser. Typhimurium. Further, B. infantis completely inhibited Salmonella-induced caspase 8 and caspase 9 activity in intestinal epithelial cells. B. infantis also reduced the basal caspase 9 and caspase 3/7 activity in epithelial cells in absence of the pathogen. Western blots and gene expression profiling of epithelial cells revealed that the decreased caspase activation was concomitant with increased phosphorylation of pro-survival protein kinase Akt, increased expression of caspase inhibiting protein cIAP, and decreased expression of genes involved in mitochondrion organization, biogenesis and reactive oxygen species metabolic processes. Hence, B. infantis exerted its protective effects by repression of mitochondrial cell death pathway which was induced in the presence of S. ser. Typhimurium.

Fusarium oxysporum (Fo) is a soil-borne fungal pathogen that causes vascular wilt disease on a broad range of plants, including agricultural crops and the model plant Arabidopsis thaliana. There are non-pathogenic members of the Fo species complex that confer defense benefits against other pathogens to the host plant, however alteration to the host’s physiology through interaction with one of these strains, Fo47, have not been described. In this study, we aimed to establish the Fo47-A. thaliana interaction and determine if Fo47 reduces disease severity of a pathogenic Fo isolate, Fo5176. Additionally, we sought to use bioinformatics to mine transcriptomic data of the infection between Fo47 and A. thaliana for putative effectors from the non-pathogenic isolate using a pipeline that is validated by identifying known effectors in the interaction between Fo5176 and A. thaliana. Phenotypic characterization of A. thaliana plants inoculated with Fo47 or Fo5176 has revealed a significant increase in rosette biomass of Fo47 inoculated plants when compared to mock (sterile water) inoculated plants. As is observed in other systems, treatment of plants with Fo47 prior to challenging with pathogenic Fo significantly reduces the disease severity over time. The results of this study suggest that Fo47 is a possible biocontrol agent against Fo5176, and that inoculation with non-pathogenic Fo alters the physiology of A. thaliana such that it has a higher rosette biomass without alterations to the water status of the plant. Our pipeline for extracting putative effectors using transcriptomic data as a critical filter generated 13 candidate genes for further experimentation to determine their role in the Fo47-A. thaliana interaction. This research reports the first known observation that Fo47 increases the shoot biomass of the host plant it is interacting with, and that the model plant A. thaliana can be used as a host to examine the spectrum of interactions capable within the Fusarium oxysporum species complex.