The largest group of plant disease resistance (R) genes that share similar structures contains a predicted nucleotide-binding site (NBS) domain. NBS domains of this class of R genes show highly conserved amino acid motifs, which makes it possible to isolate resistance gene analogs (RGAs) by PCR with degenerate primers and homology searches from public databases. Multiple combinations of degenerate primers were designed from three conserved motifs (one motif was used for a subgroup-specific primer design) in the NBS regions of R genes of various plants. All combinations of primer pairs were used to amplify genomic DNA from sorghum. TIR-specific primer combinations showed no PCR amplification in sorghum. Homology searches identified many NBS-encoding sequences among the expressed or genomic molecular database entries for sorghum. Motif analysis of the sorghum NBS sequences that were identified in this study revealed eight major conserved motifs plus two additional highly conserved motifs, but no TIR-specific motifs. Phylogenetic analysis of sorghum NBS sequences showed tree topology typical of NBS-LRR genes, including clustered nodes and longbranch lengths. Eleven distinct families of NBS sequences, representing a highly diverse sample, were isolated from Sorghum bicolor. With two exceptions, sorghum RGA families appeared to be closely related in sequence to at least one R-gene cloned from other species. In addition, deduced amino acid sequences of sorghum RGAs showed strong sequence similarity to almost all known non-TIR (Toll/Interleukin 1 Receptor)- type R-genes. Mapping with sorghum RGA markers revealed one linkage group containing four out of ten randomly selected markers, suggesting non-random distribution of NBS sequences in the sorghum genome. Rice sequences homologous to sorghum NBS sequences were found from two-way BLAST searches. Some of them were shown to be orthologs, when determined by using phylogenetic approaches which combined five different evolution models and tree-building methods.

Two different feeding trials were conducted to evaluate the effects of dietary supplementation with the dairy/yeast prebiotic GroBiotic-A (GBA) to Nile tilapia diets. A nutritionally complete basal diet was supplemented with GBA at either 1 or 2% of dry weight, and all three diets were fed to triplicate groups of juvenile fish in two consecutive trials. Trial 1 continued for 8 weeks, while Trial 2 was conducted for 5 weeks to more specifically assess immunological responses, intestinal characteristics and disease resistance of tilapia. At the conclusion of Trial 1, there were no differences in weight gain (WG) or feed efficiency (FE) among fish fed the three diets. However, fish fed the diet with GBA at 2% had significantly increased survival and noticeably elevated levels of plasma lysozyme compared to fish fed the basal diet or the diet with GBA at 1%. Similarly, at the conclusion of Trial 2, WG and FE were unaffected by GBA supplementation; however, fish fed the diet with GBA at 2% also exhibited elevated plasma lysozyme as well as significantly (P < 0.05) increased levels of extracellular superoxide anion production (EX-SOAP) by macrophages. Dendrogram analysis of denaturing gradient gel electrophoresis (DGGE) images detected a significantly different microbial community within the intestine of fish fed the diet with GBA at 2% compared to fish fed the basal diet and diet with GBA at 1%. None of the experimental diets resulted in significant improvements to survival after exposure to Streptococcus iniae due to within treatment variability. However, fish fed the diet with GBA at 2% did tend to experience reduced mortality (12.5%) as compared to fish fed the basal diet (35%). Thus, supplementation of GBA at 2% of diet did alter the gut microbiota of tilapia and enhanced immunological responses and disease resistance to S. iniae.

Atlantic salmon is a species of both scientific and economic importance, and Atlantic salmon farming is a highly profitable industry worldwide. One of the biggest challenges being faced by farms, which affects production efficiency and results in severe economic loss, is disease. In livestock production, one of the approaches taken to limit the impact of disease outbreaks is to selectively breed for improved resistance within farmed populations. Although traditional family-based resistance breeding programs have shown improvements in resistance to a variety of bacterial, viral and parasitic diseases on Atlantic salmon farms, response to selection can be slow. One way of increasing selection efficiency is through the incorporation of genetic markers into breeding programs, for marker-assisted or genomic selection. However, genomic resources for cultured aquatic species are sparse, and the generation of new and denser resources for use in selective breeding programs would be advantageous. The main focus of this thesis is the development of genomic resources in Atlantic salmon and the application of those resources to gain a better understanding of the salmon genome, particularly in the genetic basis of host resistance to infectious diseases. The first aim of this thesis was to develop improved genomic resources for Atlantic salmon, and to characterise the Atlantic salmon genome via construction and analysis of a SNP linkage map derived from RAD-Sequencing (RAD-Seq). Approximately 6,500 SNPs were assigned to 29 linkage groups, and ~1,800 male-segregating, and ~1,400 female-segregating SNPs were ordered and positioned. Overall map lengths and recombination ratios were relatively consistent between the sexes and across the linkage groups (~1:1.5, male:female). However, a substantial difference in the degree of marker clustering was seen between males and females, which is reflective of the difference in the positions of chiasmata between the two sexes. Using this map, ~4,000 Atlantic salmon reference genome contigs were assigned to a linkage group, and 112 contigs were assigned to multiple linkage groups, highlighting regions of homeology (large sections of duplicated chromosomal regions) within the salmon genome. Alignment of SNP-flanking sequences to the stickleback and rainbow trout genomes identified putative gene-associated SNPs and cross-species chromosomal orthologies, and provided evidence in support of the salmonid-specific genome duplication. In addition, based on this and other publically available RAD-Seq datasets, the utility of RAD-Seq-derived data from different species and laboratories for population genetics analyses was tested. Short RAD-Seq contigs in Atlantic salmon and nine other teleost fish were used to identify cross-species orthologous genomic relationships. Several thousands of orthologous RAD loci were identified across the species, with the number of RAD loci decreasing with evolutionary distance, as expected. Previously published broad-level relationships between orthologous chromosomes were confirmed. The identified cross-species orthologous RAD loci were used to estimate evolutionary relationships between the ten teleost fish species. Previously published relationships were recovered, suggesting that RAD-Seq data derived from different laboratories is useful for this purpose. The second aim was to characterise the genetic architecture of resistance to two viral diseases affecting Atlantic salmon production on farms: pancreas disease (PD), and infectious pancreatic necrosis (IPN). Using data and samples collected from a large population of salmon fry challenged with PD, a high heritability for resistance was estimated (h2 ~0.5), and four QTL were identified, on chromosomes 3, 4, 7 and 23. The QTL explaining the highest within-family variation for resistance was located on chromosome 3. This QTL has been confirmed in a population of post-smolts by an independent research group, highlighting the potential for its incorporation into breeding programs to improve PD resistance. For IPN, the major resistance QTL had previously been mapped to linkage group 21. However, the mutation(s) underlying this QTL effect and the consequences of these mutation(s) on the affected genes and relevant biological resistance mechanisms are unknown. To generate a list of candidate genes within the vicinity of the IPN QTL, QTL-linked DNA sequences were aligned to four model fish genomes. This identified two QTL-orthologous regions in each of the species, and gene order within these regions was highly conserved across species. Analysis of gene expression patterns between IPN resistant and susceptible salmon in a viral challenge experiment revealed that the five most significantly differentially-expressed genes mapped to the QTL-orthologous region on linkage group II of stickleback. Pathway enrichment analysis across all differentially-expressed genes suggests that biological pathways influencing viral infection stress response/entry/replication, cellular energy production and apoptosis may be involved in resistance during the initial stages of IPN virus (IPNV) infection. These results have provided the basis for further study of the putative involvement of these candidate genes and pathways in genetic resistance to IPNV. In summary, the results and resources presented in this thesis extend our current understanding of the salmon genome and the genetic basis of resistance to two viral diseases, and provide resources with the potential to be used in Atlantic salmon selective breeding programs to tackle disease outbreaks.

<p>Drug-delivery nano-systems enhances the potency of bioactive molecules due to its increase membrane permeability, as a result of their sub-cellular size. The concept of engineered nano-carriers may be a promising route to address confounding challenges in agriculture that could lead to an increase in crop production while reducing the environmental impact associated with crop protection and food production. A key motivation of this work is to evaluate the potential use of drug delivery nanosystems in agriculture, especially in the area of disease control. To this end, identifying the most suitable materials to serve as carrier and cargo is imperative. Understanding their bioactive properties and their physical-chemical characteristics is critical because these influences not only their biological effects on plants and environmental impact, but also, the fabrication process and potential scaling-up, enabling practical and relevant field applications in the future. </p> <p> </p> <p>In this work, chitosan was selected as nano-carrier material because of its biological and chemical properties. The chemical structure of chitosan allows spontaneous assemble of core-shell like nanostructures via ionic gelation, has enabled it to be used as nano-carrier biomaterial intended for delivery of bioactive cargo. In agriculture, the use of chitosan is of special interest due to its immune-modulatory activity elicited in plants. However, due to its inherent molecular heterogenicity, the formulation and fabrication of stable and low inter-batch variability chitosan nanocarriers via ionic gelation is difficult and time consuming.</p> <p> </p> <p>A myriad of different bioactive molecules has been tested as payload, encapsulated into chitosan-based delivery nano-systems for a range of purposes ranging from biomedicine, pharmaceutical, food and agriculture. In this work plant derived essential oils were selected as bioactive payload. Essential oils are at the core of the plant communication process with their phytobiome, including plant pathogens. Molecules from essential oils can carry an air-borne message serving as a plant-to-plant communication system (a phenomenon known as allelopathy) that activate the plant defense mechanisms. Encapsulation of essential oils into chitosan nanocarriers is only possible by forming nano-emulsions. </p> <p>Despite the potential benefits from the use of chitosan and essential oils in agriculture, its use at a large scale has been hindered by the overwhelming inconsistencies in the current literature, regarding their formulation and fabrication. This work addresses these problems and presents evidence that support the feasibility of producing highly chitosan nanocarriers loaded with essential oils, in a facile and rapid way, using FDA-grade materials only, without the need of expensive or specialized instrumentation. </p> <p> </p> <p>The plant-pathogen compatible interaction between <i>A. thaliana</i> and <i>B. cinerea</i> was used as biological model to test the hypothesis that chitosan nano-carriers and essential oil nano-emulsions can enhance the quantitative disease resistance of plants against broad host-range necrotrophic pathogens. We found that these treatments display a dose-dependent response in plants triggering a systemic immune response. Image-based phenotyping analysis showed that chitosan nanoparticles alone, as well as loaded with d-limonene, significantly enhanced the disease resistance of <i>A. thaliana</i> against <i>B. cinerea</i>. Nano-emulsions using essential oils from cinnamon, clove, coriander and red thyme also produced similar effects on the defense response in the pathosystem under study. Functional analysis of the differentially expressed genes from treated plants revealed that these treatments up-regulated the biological process involved in “stress management”, while down-regulated the biological process required for normal growth and development during ideal, non-stressful conditions.</p>

Heterodera glycines, soybean cyst nematode (SCN) causes more than one billion dollars soyben production loss in the U.S. annually. SCN is an obligate parasite of specialized feeding cells within the host root known as syncytium. The SCN resistance genes and signaling pathways in soybean have not been fully characterized. Gene expression analysis in syncytium from compatible and incompatible interactions identified candidate genes that might involve conferring resistance to SCN. This dissertation aimed to investigate the biological functions of the candidate resistance genes to confirm the roles of these genes in resistance to SCN. The study demonstrated a role of syntaxin 31-like genes (Gm-SYP38) in resistance to SCN. Overexpression of Gm-SYP38 induced the transcriptional activity of the cytoplasmic receptor-like kinase BOTRYTIS INDUCED KINASE 1 (Gm-BIK1-6). Overexpression of Gm-BIK1-6 rescued the resistant phenotype. In contrast, Gm-BIK1-6 RNAi increased parasitism. In another experiment, the expression of a Glycine max homolog of LESION SIMULATING DISEASE1 (LSD1) resulted in the transcriptional activation of ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and NONEXPRESSOR OF PR1 (NPR1), that function in salicylic acid (SA) signaling, implicating the involvement of the antiapoptotic, environmental response gene LESION SIMULATING DISEASE1 (LSD1) in defense that is demonstrated here. The study also investigated the role of SNARE components (genes functioning in membrane fusion) in resistance to SCN. Experiments showed that SNARE functions in concert with a beta-glucosidase having homology to PEN2 and an ATP binding cassette transporter having homology to PEN3. This study provides novel information for the genetic improvement of soybean for enhanced disease resistance.

No description available.

This research was conducted to analyze the genetics of soybean mosaic virus (SMV) resistance in soybean [Glycine max (L.) Merr.] and to determine allelic relationships of SMV resistance genes and their interactions with SMV strain groups. In the first part of this study, the inheritance of SMV resistance in OX670 and 'Harosoy' was studied to determine the source and identity of the resistance gene/genes in OX670. Other researchers reported that OX670 possesses a single gene at a locus independent of Rsv1 and assigned the gene symbol Rsv2. Rsv2 was presumably derived from the cultivar 'Raiden'. However, later work showed that Raiden contains a single resistance gene at the Rsv1 locus, raising the possibility that the resistance gene in OX670 was not from Raiden. Harosoy and its derivatives make up much of the remaining pedigree of OX670. Results from crosses of OX670 with susceptible cultivars indicate that it contains two independent genes for SMV resistance. One is allelic to the Rsv1 locus, expresses resistance to SMV-G1 and G7 and is derived from Raiden. The other is allelic to the Rsv3 locus, expresses resistance to SMV-G7 but susceptibility to SMV-G1 and is derived from Harosoy. Therefore the Rsv2 locus does not appear to exist in OX670 or its ancestors. The presence of Rsv1 and Rsv3 makes OX670 resistant to all SMV strains from G1 through G7. The second study was conducted to investigate the inheritance and allelomorphic relationships of resistance gene(s) in 'Tousan 140' and 'Hourei', which were reported to carry single independent resistance genes when inoculated with the Japanese SMV strain C. Both of these lines exhibit resistance to strains SMV-G1 through G7. This inheritance study shows that Tousan 140 and Hourei each possess two resistance genes. One of the genes in Hourei confers resistance to SMV-G1 and G7 strains; the other gene confers susceptibility to SMV-G1 but resistance to SMV-G7. Allelism tests indicate that one of the genes in both Hourei and Tousan 140 is allelic to Rsv1, and the other is allelic to Rsv3. The two genes in Tousan 140 were separated into individual lines, R1 and R2. R1, most probably containing Rsv1, exhibited resistance to SMV-G1 through G3 but was susceptible to SMV-G5 through G7. Line R2, most likely possesses Rsv3 gene, was susceptible to SMV-G1 through G3 but resistant to SMV-G5 through G7. Therefore, presence of these two genes makes Tousan 140 resistant to SMV-G1 through G7. The objective of the third study was to investigate inheritance and allelomorphic relationships of SMV resistance in PI88788. PI88788 exhibits resistance to SMV-G1 through G7. Genetic analysis of our data reveals that SMV resistance in PI88788 is conferred by a single gene at a locus tentatively labeled 'Rsv4'. Expression of this gene in the homozygous state decreased accumulation rate and prevented vascular movement of SMV. In the heterozygous state vascular movement of the SMV was delayed but not prevented. / Ph. D.

BACKGROUND:The rhizome, the original stem of land plants, enables species to invade new territory and is a critical component of perenniality, especially in grasses. Red rice (Oryza longistaminata) is a perennial wild rice species with many valuable traits that could be used to improve cultivated rice cultivars, including rhizomatousness, disease resistance and drought tolerance. Despite these features, little is known about the molecular mechanisms that contribute to rhizome growth, development and function in this plant.RESULTS:We used an integrated approach to compare the transcriptome, proteome and metabolome of the rhizome to other tissues of red rice. 116 Gb of transcriptome sequence was obtained from various tissues and used to identify rhizome-specific and preferentially expressed genes, including transcription factors and hormone metabolism and stress response-related genes. Proteomics and metabolomics approaches identified 41 proteins and more than 100 primary metabolites and plant hormones with rhizome preferential accumulation. Of particular interest was the identification of a large number of gene transcripts from Magnaportha oryzae, the fungus that causes rice blast disease in cultivated rice, even though the red rice plants showed no sign of disease.CONCLUSIONS:A significant set of genes, proteins and metabolites appear to be specifically or preferentially expressed in the rhizome of O. longistaminata. The presence of M. oryzae gene transcripts at a high level in apparently healthy plants suggests that red rice is resistant to this pathogen, and may be able to provide genes to cultivated rice that will enable resistance to rice blast disease.

Master of Science / Genetics Interdepartmental Program - Plant Pathology / Frank F. White / Sanzhen Liu / Plants and bacterial pathogens are in constant co-evolution to survive and sustain the next generation. Plants have two well-characterized levels of active defense -pathogens-associated molecular patterns (PAMPs)-triggered immunity (PTI) and effectors-triggered immunity (ETI). Some plants that are hosts for bacterial pathogens employing type three secretion system transcription activator-like (TAL) effectors have evolved a unique form of ETI, namely TAL effector-mediated ETI. TAL effectors induce expression of specific disease susceptibility (S) genes. Rice and pepper have evolved resistance genes termed terminator (T) genes, which have promoters that bind TAL effectors and, upon expression of the T gene, elicit a hypersensitive reaction (HR) and cell death. Only five T genes have been cloned, and the origin of most T genes is unknown. To determine the presence of candidate T genes in other plants species, a bioinformatics-based mining was designed. The basic approach utilized three structural features common to four terminator genes: a short trans-membrane domain, a secretion signal domain, and a length of <200 amino acid residues. Soybean was chosen as the test plant species, and 161 genes were retrieved that fulfilled the three parameters using R and Perl software programs. Further, functional annotation of candidate genes was conducted by comparisons to genes in public databases. Major classes of proteins found included unique and hypothetical, defense/stress/oxidative stress associated, DNA-binding, kinases, transferases, hydrolases, effector-related tRNA splicing, and F- box domain proteins. The potential T genes will serve as candidates for experimental validation and new resources for durable resistance strategies in crop species.

O objetivo deste trabalho foi avaliar a resistência parcial e a tolerância à ferrugem asiática, em sete cultivares comerciais e três linhagens de soja. Os ensaios foram conduzidos no município de Jataí, GO, no ano agrícola de 2005/2006. Ensaios distintos foram realizados para avaliação da resistência parcial e para avaliação da tolerância, todos repetidos em três épocas de plantio, a fim de se obter intensidade variável da doença em diferentes estádios fenológicos da cultura. Nos ensaios para avaliação da resistência parcial, o delineamento experimental foi o de blocos cazualizados com cinco repetições e cada parcela experimental foi composta por cinco linhas de seis metros de comprimento. A severidade da doença foi estimada em intervalos semanais, a partir do surgimento dos primeiros sintomas até a desfolha completa. Os dados foram analisados por meio de regressão não linear e o modelo logístico foi o que apresentou melhor ajuste. As curvas de progresso da doença para os cultivares EMGOPA-315, Luziânia, Pintado, Conquista, COODETEC-219 e para as linhagens 1, 2, e, 3 não apresentaram estabilização assintótica da severidade em função da desfolha precoce, ficando evidente apenas a fase exponencial de crescimento da doença. As epidemias apresentaram comportamento explosivo e foram muito semelhantes, demonstrando que esses genótipos foram igualmente susceptíveis ao patógeno. Os cultivares Tianá e EMGOPA-313 foram os únicos que apresentaram estabilização assintótica da severidade, em níveis muito abaixo de 1, o que evidenciou a presença de resistência parcial nesses cultivares. Nos ensaios para avaliação da tolerância, o delineamento experimental foi o de blocos casualizados com cinco repetições, com delineamento dos tratamentos em parcelas subdivididas. As parcelas (5 linhas de 12 m de comprimento) foram compostas pelos genótipos, e as subparcelas (5 linhas de 6 m de comprimento), pelo tratamento ou não com fungicida tebuconazole (0,5 L.ha-1) para controle da ferrugem. A severidade da doença foi estimada uma única vez e a tolerância foi avaliada pela análise estatística da diferença de produtividade, peso de mil grãos e de número de vagens e grãos por planta, entre subparcelas tratadas e não tratadas com fungicida, em cada época de plantio. Nenhum genótipo avaliado mostrou-se tolerante à ferrugem asiática da soja. / This work aimed the evaluation of partial resistance and tolerance to soybean asian rust on seven soybean genotypes and 3 lines. The research was conducted at Jataí, GO, during the growing season of 2005/2006. Experiments for partial resistance evaluation were carried out separately from experiments to evaluate tolerance, and in order to have different levels of disease severity, on different soybean growth stages, three trials for each experiment were carried out, each one with a different planting date during the season. Experiments for partial resistance evaluation were arranged on a randomized block design and each experimental unit was made by five soybean lines, 6 m long. The disease severity was estimate on week intervals from the first pustules appearance to the total defoliation. Analyses were done by non linear regression and the logistic model presented the best fitness to the observed data. The early defoliation on cultivars EMGOPA-315, Luziânia, Pintado, Conquista, COODETEC-219 and on lines 1, 2, e, 3 did not allowed the disease progress curves to reach asymptotical stabilization and for such genotypes, just the exponential part of the disease growth could be seen. Epidemics presented an explosive growth and the disease progress curves for such genotypes were similar, suggesting that they were similarly susceptible to the pathogen. The cultivars Tianá e E-313 were the only ones that presented asymptotical stabilization of disease and that happened on severity levels much smaller than 1, what was considered an evidence of partial resistance on those cultivars. Experiments conducted for tolerance evaluation were arranged in a split plot design with five replicates. The main plots (5 soybean rows, 12 m long) were composed by the soybean genotypes and the subplots (5 soybean rows, 6 m long) were those with or without fungicide (tebuconazole 500 mL.ha-1) application for rust control. Disease severity was estimate only once and tolerance was evaluated by the statistical analyses of difference of yield, thousand grain weight and pod and grain numbers, between treated and untreated subplots. None soybean genotypes analyzed presented tolerance to soybean asian rust.