Doctor of Philosophy / Genetics Interdepartmental Program / Scot H. Hulbert / Frank F. White / Many maize lines carry Rxo1, an NB-LRR gene that confers a rapid hypersensitive response (HR) after infiltration with the rice streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc) or the maize stripe pathogen Burkholderia andropogonis (Ba) carrying the effector genes avrRxo1 or avrRba1 respectively. Interestingly, when expressed as a transgene in rice, Rxo1 also confers a strong and rapid HR to Xoc strains harboring the avrRxo1 type III effector gene. To gain insights into the Rxo1 signaling network, we used a combination of functional genomics and bioinformatics, molecular genetics and reverse genetics. Microarray experiments were carried out to investigate the temporal expression profiles of nonhost and host responses to isogenic strains of Xanthomonas oryzae pv. oryzae (Xoo), the rice bacterial blight pathogen, and Ba with and without the Xoc type III secreted effector gene avrRxo1. Xoc AvrRxo1 induces disease resistance in maize when delivered by Xoo or Ba. We show that recognition of the two bacterial pathogens is translated into similar transcriptional outputs. Cluster analyses revealed that Xoo and Ba co-regulated genes display different kinetics and amplitudes and showed that gene clusters are associated with overrepresentation of known and putative novel DNA cis regulatory elements. One early induced gene, ZmPti1b, is a serine threonine kinase. RNAi-mediated gene silencing of a rice ortholog of ZmPti1b, OsPti1a, revealed that OsPti1a is required for Rxo1-governed resistance. Using a full length coding sequence as bait to screen a yeast-two-hybrid library, we identified 11 rice proteins that interact with RXO1. Functional analysis of two showed that Os1PVOZ, encoding a putative transcription factor, is required for Rxo1-dependent HR whereas OsATL6, a putative RING finger type E3 ubiquitin ligase gene is dispensable. Scanning of the rice genome for putative DNA binding sites suggests that Os1PVOZ is a master regulator of many signal transduction pathways, including those that mediate plant defense responses. Our investigations identified key signaling components that mediate Rxo1-specified resistance and possibly resistance mediated by other R genes.

Doctor of Philosophy / Department of Plant Pathology / Jan E. Leach / Both qualitative and quantitative resistance mechanisms are important contributors to disease resistance in rice. To identify useful sources of durable resistance for Korean breeding programs, the distribution of rice blast isolates compatible to widely used resistance (R) genes was analyzed. Interactions of 3,747 Korean rice Magnaporthe oryzae isolates with eight monogenic lines, each harboring a major blast R gene, were tested. Lines with R gene Pi-9 and Pi-5 were susceptible to the fewest M. oryzae isolates, and therefore, this gene might be applied for blast resistance in breeding programs in Korea. Six major blast resistance genes were susceptible to more than 60 % of the population, suggesting limited utility of these genes in breeding programs. Quantitative trait loci (QTL)-based resistance is predicted to provide durable and broad spectrum resistance to rice diseases. A candidate gene approach was applied to a population of 164 recombinant inbred lines to identify sources of quantitative resistance. Resistance gene analogs and defense response genes were mapped on the rice chromosomes, and analyzed for their association with blast and bacterial blight resistance QTL. A total of 21 putative QTL for blast resistance were identified on chromosomes 1, 4, 5, 6, 8, 9 and 12. Four putative QTL for bacterial blight resistance were identified on chromosome 3, 5 and 10. Thirteen RGA markers were associated with 11 different QTL on chromosome 1, 5, 8, and 9. The role of one disease resistance QTL associated gene, Os02g39330, encoding a chitinase was investigated for contributing to basal defense responses. RNAi silencing was used to evaluate contributions of the gene for the resistance to Rhizoctonia solani and M. oryzae. Five transgenic lines harboring the silencing construct and which differed in the level of expression of Os02g39330 were screened for responses to R. solani and M. oryzae. The chitinase gene expression levels were inversely correlated with sheath blight disease severity, suggesting a role for this defense gene in resistance to R. solani. Rice blast disease was not affected by silencing Os02g39330. Both qualitative and QTL-based resistances provide valuable sources of disease resistance, and a combination of R gene Pi-9 and QTL harboring the Os02g39330 chitinase may help to stabilize resistance.

Doctor of Philosophy / Department of Plant Pathology / William W. Bockus / Septoria tritici blotch caused by Mycosphaerella graminicola (anamorph Septoria tritici), is an important disease of wheat worldwide capable of reducing yields by as much as 30 to 40%. In Kansas, the disease is widespread and losses in individual fields can exceed 25%. This study examined the genetic structure of Kansas populations of M. graminicola at different spatial scales (micro-plot, macro-plot, and statewide) using amplified fragment length polymorphism (AFLP) markers. Three primer pairs were used to resolve 174 polymorphic loci from 476 isolates. The results indicated high levels of genotypic variability, which is consistent with a genetically diverse initial inoculum. Genetic identities among populations representing the three spatial scales were >98%. Tests for differentiation among populations due to population subdivision revealed that on average 97.5% of the genetic variability occurred within populations with a correspondingly high migration rate of 16 to 23 individuals per generation. We observed little evidence of linkage disequilibrium, on average, only 4.6% of locus pairs were in disequilibrium. Our results indicate that Kansas populations of M. graminicola are characterized by regular recombination, are genetically diverse, and appear to be homogenous across different spatial scales. These populations are probably components of a larger pathogen pool that is distributed at least across much of Kansas and probably the central Great Plains. Because of the frequent recombination, the risk of adaptation of Kansas populations of M. graminicola to fungicide treatments or resistance genes is high and could be dispersed very quickly, whether these new pathogenic traits occur locally through mutation or by migration from other areas.

Master of Science / Department of Plant Pathology / John P. Fellers / Harold N. Trick / Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV), are two of the major viruses in the Great Plains of the United States. Cultural practices and mite vector control are the primary methods of disease management; however, they are not fully effective. Resistant varieties are also deployed, although some of the lines present temperature sensitive resistance or negative agronomic properties are linked to resistance. Alternative approaches to viral resistance are needed. RNAi has been shown to play a role in viral defense response and has been successfully used as a biotechnological tool to preprogram viral resistance in transgenic plants. RNAi reduces the expression of specific genes by targeting the accumulation of mRNA. The mechanism is activated by the presence of dsRNA, which is processed into small non-coding sequence that serves as a guide for degrading RNA in a sequence specific manner. In this work, the RNAi approach was used to elicit resistance against WSMV and TriMV. Immature embryos of the wheat cv "Bobwhite" were independently co-transformed with pAHC20, containing the bar gene for glufosinate selection, and either the hairpin construct targeting the coat protein of WSMV or TriMV. After tissue culture, PCR was used to determined the presence of the RNAi CP transgene in putative transformed plants. Eight WSMV and ten TriMV CP RNAi transgenic plants were obtained from the bombardment experiments. Transgenic T1 and T2 seeds were collected and transgene expression was established through RT-PCR. In order to determine viral resistance, T1 and T2 progeny was mechanically inoculated. ELISA results indicated a differential resistance response among the tillers evaluated in each line in T1 generation for both WSMV and TriMV constructs. In T1 generation resistance was seen in up to 60% of the plants evaluated for both constructs, although some events that showed transgene presence did not exhibited resistant phenotype. Analyses of transgene presence and expression in T2 generation evidenced events of transgene silencing and deletion. Regardless of these phenomena, consistent resistance response in two lines of WSMV CP construct and one TriMV CP transgenic line was found.

Doctor of Philosophy / Department of Plant Pathology / Bernd R. Friebe / Hexaploid wheat (Triticum aestivum L., 2n=6x=42, AABBDD) is a recent polyploid and originates from a limited number of founder genotypes. Domestication bottlenecks further reduced genetic diversity. The wheat gene pool, which consists of landraces and wild relatives such as rye (Secale cereale L.), Leymus racemosus Tien and Elymus tsukushiensis Honda, is a rich source of genetic diversity. Agronomically important traits can be transferred from these gene pools to wheat through chromosome engineering. This dissertation describes chromosome engineering and pre-breeding efforts for resistance to Hessian fly and Fusarium head blight (FHB) in wheat. The germplasm with a whole-arm rye translocation, T2BS.2R#2L, contains the highly effective Hessian fly resistance gene, H21, and an unnamed powdery mildew resistance gene. Directed chromosome engineering was used to shorten the whole-arm rye segment. The recovered wheat-rye recombinant chromosome, T2BS.2BL-2R#2L, had a shorter rye segment but still contained the H21 gene and was transferred through backcrosses to adapted winter and spring wheat cultivars. This study released the germplasm KS09WGGRC51, which is used in wheat breeding programs in the U.S.A. Two novel sources of FHB resistance were identified in L. racemosus and E. tsukushiensis. Fhb3 present in the wheat-L. racemosus T7AL.7Lr#1S Robertsonian translocation was transferred into the adapted Kansas winter wheat cultivar Fuller. The wheat-E. tsukushiensis disomic addition translocation line confers FHB resistance. Ph1b-induced homoeologous recombination was used to produce wheat-E. tsukushiensis recombinants. The distal and interstitial recombinants were identified using molecular markers and genomic in situ hybridization (GISH). A combination of molecular cytogenetic analyses determined that the distal recombinant involved wheat chromosome 1A and a small distal segment originating from the E. tsukushiensis chromosome arm, 1E[superscript]ts#1S, resulting in the recombinant chromosome T1AL.1AS-1E[superscript]ts#1S. The interstitial recombinant involves an unidentified wheat chromosome and appears to be highly rearranged. Both recombinants confer high levels of type II FHB resistance (resistance to spread within the head) based on point inoculations in the greenhouse. To date, these two recombinants are the smallest alien introgression with FHB resistance in common wheat. This germplasm material has been released as KS14WGRC61. The distal recombinant can be used directly for breeding of FHB-resistant cultivars worldwide.

Master of Science / Department of Plant Pathology / Christopher R. Little / Christopher R. Little / Charcoal rot, caused by Macrophomina phaseolina, is the most important soybean disease in Kansas. Several strategies have been recommended to control this disease including crop rotation, lower plant densities, biological control, plant resistance and tolerance, and fungicide application. However, those techniques have not been completely effective and the information concerning soil texture, irrigation and micronutrient fertility (particularly manganese) upon charcoal rot disease severity and the pathogen population is limited. The objective of this study was to determine key factors that affect the biology of M. phaseolina and charcoal rot processes under laboratory, greenhouse and field conditions. M. phaseolina microsclerotia were produced from PDA pure isolate and infested Japanese millet in the laboratory and characterized by different techniques such as serial dilutions in semi selective media with the aim to produce quality inoculum to reliably infect soybean seedling roots under greenhouse conditions; production of inoculum by infesting Japanese millet was the most efficient method. Root colonization and root infection of soybean seedlings was assessed through the use of M. phaseolina inoculum under controlled conditions in the greenhouse. Root infection by M. phaseolina and microsclerotia longevity in soil is determined by environmental factors such as soil moisture content, soil texture and source of inoculum. The objective of the greenhouse study was to determine the impact of these variables on seedling root infection at the V1 and V2 development stages. Artificial soils with different textures were infested; M. phaseolina microsclerotia and soybean seedlings were exposed to different soil moisture contents including pot saturation, pot (field) capacity, and permanent wilting point. Soil populations and levels of root colonization for the stages were assessed by estimating CFUs and root length. Results indicate that soil texture has a significant impact upon root morphology and root length. Root populations of M. phaseolina were significantly higher in sandy soil textures and lower in the fine-textured soils, suggesting an impact of soil water holding capacity in the root infection process. The effect of water stress on seedling root colonization by M. phaseolina indicates that early infection may be more important than previously thought. A field study was also conducted to determine the effect of the aforementioned variables in a 2-year field experiment conducted at two Kansas locations. Pathogen colonization was iii assessed by measuring colony-forming units (CFUs) from ground root tissue at R2-R4 (post-flowering/early pod development) and R8 (maturity) stages. Soil populations (pre-planting and post-harvest) of M. phaseolina, yield parameters, and plant characteristics were obtained. Results indicated that there are complex relationships between soil physiochemical properties (pH, NPK content, exchangeable cations, and organic matter) and soil texture (sand, soil, and clay composition), which may mitigate disease severity and pathogen levels in host tissue. Results also indicated that in natural M. phaseolina-infested soils, cropping history and soil texture play an important role in charcoal rot processes and influence the levels of pathogen soil populations, root colonization at maturity and, more importantly, soybean yield.

Doctor of Philosophy / Department of Plant Pathology / Jan E. Leach / Because it is predicted to be durable and broad spectrum, quantitative trait loci (QTL)-based resistance is an important option for rice disease control. However, manipulation of this type of resistance requires knowledge of the contributing genes. This study demonstrates the contribution of two of three defense response (DR) genes to QTL-governed resistance, and identifies a third gene that negatively regulates resistance. The contribution to QTL-governed resistance of one of nine rice OsPAL genes, which encode phenylalanine ammonia-lyase, was determined using reverse genetics. Mutant ospal4 contains a 750 bp deletion in OsPAL4 and was identified using a PCR-pooling strategy. OsPAL4 underlies a QTL on chromosome 2, and is located in cluster with three other OsPAL members. Rice lines mutated in OsPAL4 are more susceptible to a virulent strain of Xanthomonas oryzae pv. oryzae (Xoo) than lines with the wild type allele. RNAi suppression was used to evaluate the contributions of genes encoding oxalate oxidase-like proteins (OsOXL) and a 14-3-3 protein (GF14-e) to disease resistance. Silencing of 12 OsOXL genes clustered on chromosome 8, varied from suppression of a few gene members to silencing of all expressed family members. Screening of transgenic lines by challenge with Magnaporthe grisea (Mg), the rice blast pathogen, revealed that the more chromosome 8 OsOXL genes suppressed, the more susceptible the plants were to Mg. GF14-e co-localizes with a disease resistance QTL on chromosome 2. Specific suppression of GF14-e by RNAi silencing did not result in enhanced susceptibility to Mg. Instead, the lines exhibited spontaneous HR-type lesions. The presence of this lesion mimic phenotype correlated with enhanced resistance to a virulent strain of Xoo, suggesting that the GF14-e encoded 14-3-3 protein functions as a negative regulator of plant cell death and bacterial resistance in rice. This study supports the hypothesis that DR genes, such as OsOXL and OsPAL4 contribute to disease resistance governed by QTL. The role of GF14-e is less clear, however its down regulation may contribute to QTL-governed resistance. Thus, incorporation of regions harboring the effective DR gene alleles into rice will enhance broad spectrum and durable resistance.

Doctor of Philosophy / Department of Plant Pathology / Barbara S. Valent / The hemibiotrophic rice blast fungus Magnaporthe oryzae undergoes complex morphological development throughout its infection cycle. From 8-20 hours after a fungal spore lands on a leaf surface, the fungus differentiates a complex appressorium that punctures the host cuticle. By ~24 hours post inoculation (hpi), the fungus grows inside an epidermal cell as a primary hypha, and by 36 hpi the fungus has differentiated specialized biotrophic invasive hyphae (IH) that are filling the first-invaded cell and moving into neighbor cells. Throughout its life cycle, IH invade living rice cells although invaded cells appear dead when the fungus moves into the next cell. Biotrophic invasion must be mediated by fungal effectors, proteins that pathogens secrete inside live host cells to control them. However, little is known about blast effectors, and the low fungal biomass in early infection stages complicates identification of effector genes, as well as identification of rice genes controlled by effectors. The characterized AVR-Pita effector gene is specifically expressed in planta, but it was not clear how its gene expression pattern changed in different infection stages. We found that AVR-Pita is first expressed around the time of penetration. AVR-Pita is highly expressed in IH developing in asymptomatic tissue from 36 hpi to as late as 7 days post inoculation when lesions are maturing. Using inoculated rice sheaths, we successfully enriched for infected tissue RNA that contained ~20% IH RNA at 36 hpi. We compared IH gene expression to expression in mycelium from pure culture using a whole-genome M. oryzae oligoarray, and we compared infected rice gene expression to expression in mock-inoculated tissue using a rice oligoarray. Rice genes that were induced >50-fold during infection were enriched for genes involved in transferring information from sensors to cellular responses. Fungal genes that were induced >50-fold in IH included known effectors and many IH-specific genes encoding hypothetical secreted proteins that are candidate effectors. Gene knock-out analyses of three putative effector genes failed to show major effects on pathogenicity. Details of the blast interaction transcriptome will provide insights on the mechanisms of biotrophic plant disease.

Master of Science / Department of Electrical and Computer Engineering / Karen A. Garrett / Caterina M. Scoglio / With rapid global movement of epidemics, research efforts to characterize dynamics of epidemics have gained much focus. Traditional epidemiological models have focused on only temporal components of epidemics. Development of spatio-temporal models proved to be a notable achievement in epidemiology. Network-based epidemiological models enable better handling of spatial and temporal components of an epidemic. Early network models considered a binary level of contact between infected entities, which is an idealistic approach. A realistic approach would use weighted edges which signify the level of interaction between the nodes where the edge-weights change over time as a function of environmental factors. Estimation of edge weights from observed time series is a relatively less explored area for network modeling. Dynamic networks make the problem more complicated as edge weights change over time. Estimation of parameters for models describing the edge weights as a function of variables that change in time has the potential to provide better general models. Soybean rust (caused by Phakopsora pachyrhizi) is an important disease globally and its occurrence in the US has been studied extensively since its introduction in 2004. Rust is a fungal disease which propagates as a result of the fungal spores being carried by the wind. In this thesis, a network network based model is proposed to predict the intensity of spread of the disease in space and time. This model uses the host abundance and wind data and the observed rust incidence time series to compute the edge-weights. Also, the edge-weights in the model change over time thus following a dynamic approach. In order to cut costs involved with the establishment and maintenance of infection monitoring sites, the effect of removal of monitoring nodes using various strategies has also been analyzed in this thesis. The model has been tested with observed soybean rust data from sentinel plot network from across the United States.

Doctor of Philosophy / Department of Plant Pathology / John P. Fellers / Harold Trick / In Kansas, wheat (Triticum aestivum L.) is severely affected by the biotrophic fungus Puccinia triticina (leaf rust). Although resistant varieties have been developed, the fungus tends to overcome new sources resistance very quickly. Plants have evolved a single gene (R genes) defense network that can recognize specific pathogen effectors (Avr), in a gene-for-gene manor. In rusts, effectors are secreted proteins responsible for inducing the uptake of nutrients and inhibit host defense responses. Identification of secreted proteins during the infection may help to understand the mode of infection of P. triticina. Little is known about molecular interactions in the pathosystem wheat-leaf rust and no Avr genes from cereal rusts have been cloned. In order to understand pathogenicity in leaf rust and generate new alternatives for disease control, the goal of this research is identify P. triticina secreted proteins from a collection of expressed genes during the infection, and to characterize putative Avr function for three candidates. From 432 EST’s derived from haustoria and infected plants, fifteen secreted proteins were identified and 10 were selected as potential avirulence candidates. Pt3 and Pt 51 are two P. triticina (Pt) candidates expressed specifically in the haustoria and encode small cysteine-rich secreted proteins. Eight candidates are expressed at early stages of infection, during spore germination and 6 days after inoculation. They are small-secreted proteins. None are repetitive elements or have nuclear localization signals. They also do not share a conserved motif with known filamentous fungus Avr proteins. Five candidates are novel proteins, two have similarity with predicted proteins, one is homologous with Hesp-379-like protein, one is homologous with superoxide dismutase, and one has a cell glucanase predicted function. Pt3, Pt12 and Pt27 were tested by transient expression experiments using co-bombardment with GUS into leaf rust resistant isogenic lines. Reduction in the expression of reporter gene GUS co-expressed with Pt27 indicates a potential avirulence factor for Lr26 in wheat.