Doctor of Philosophy / Genetics Interdepartmental Program - Plant Pathology / Bikram Gill / The diploid D genome species, Aegilops tauschii Coss. (2n=2x=14,DD) has provided numerous genes for resistance to diseases and insect pests that attack bread wheat (Triticum aestivum L. (2n=6x=42, AABBDD). Wheat production is currently threatened by broadly virulent races of the ‘Ug99’ lineage of wheat stem rust caused by the fungus, Puccinia graminis f.sp. tritici Pers. & Eriks. Screening of a large set of Ae. tauschii germplasm for resistance to TTKSK (Ug99) identified potentially novel sources of resistance. To expedite utilization of TTKSK resistance from Ae. tauschii, a direct hybridization approach was established that integrates gene transfer, mapping and introgression into one process. Direct crossing of Ae. tauschii accessions with an elite wheat breeding was used to initiate transfer of resistance. Genetic mapping of resistance was accomplished during gene transfer through development of BC[subscript]2 mapping populations. Bulked segregant analysis of BC[subscript]2F[subscript]1 genotypes at 70 SSR loci across the D genome identified the chromosome locations of stem rust resistance genes and facilitated genetic mapping. Using this approach, TTKSK resistance from CDL4424 and TA1662 was mapped on chromosome arm 1DS flanked distally by Xwmc432 and proximally by Xwmc222 at 4.4 cM, TA10187 on 6DS linked to Xcfd49 at 1.9 cM and TA10171 on 7DS linked Xwmc827 at 0.9 cM. TTKSK resistance from additional Ae. tauschii accessions CDL4366, TA1615, TA1642, TA1693 and TA1718 has been recovered in segregating populations but await mapping. Altogether, TTKSK resistance from eight Ae. tauschii accessions has been introgressed to a hard winter wheat genetic background. Three new stem resistance genes have been tagged with molecular markers for marker assisted breeding and will provide valuable material for stem rust resistance breeding and gene pyramids for effective control of stem rust.

The recently-emerged field of chemical genomics is centered on the use of small molecules to perturb biological systems as a means of investigating their function. In order to employ this approach for the study of plant-pathogen interactions, I established an assay in which Arabidopsis thaliana seedlings are grown in liquid media in 96-well plates. Inoculation of these seedlings with a virulent strain of the bacterial phytopathogen Pseudomonas syringae resulted in macroscopic bleaching of the cotyledons of these seedlings. This symptom was used as the basis for high-throughput chemical genomic screens aimed at identifying small molecules that protect Arabidopsis seedlings from infection. One of the first chemicals identified through this screen was the sulfanilamide compound sulfamethoxazole (Smex). This compound was later shown to also reduce the susceptibility of both Arabidopsis and wheat to infection by the fungal pathogen Fusarium graminearum, suggesting a broad spectrum of activity. More detailed investigations of Smex indicated that the protective activity of this compound did not derive from antimicrobial effects, and that this activity was not executed through common defence-related signalling pathways. The folate biosynthetic pathway enzyme dihydropteroate synthase is a known target of sulfanilamides, and it does appear to contribute to Smex-induced disease resistance, albeit in a folate-independent manner. In order to identify downstream mediators of Smex activity, I initiated two forward genetic screens intended to recover mutants with altered sensitivity to Smex in a seedling growth assay. Interestingly, while these screens yielded mutants with striking Smex sensitivity phenotypes, disease resistance phenotypes were not altered. Gene expression profiling of Arabidopsis tissues treated with Smex prior to bacterial inoculation suggested that this compound generally affects lipid signalling. Altogether, it is evident that Smex elicits a complex set of responses in Arabidopsis with apparently non-overlapping phenotypic outputs.

The recently-emerged field of chemical genomics is centered on the use of small molecules to perturb biological systems as a means of investigating their function. In order to employ this approach for the study of plant-pathogen interactions, I established an assay in which Arabidopsis thaliana seedlings are grown in liquid media in 96-well plates. Inoculation of these seedlings with a virulent strain of the bacterial phytopathogen Pseudomonas syringae resulted in macroscopic bleaching of the cotyledons of these seedlings. This symptom was used as the basis for high-throughput chemical genomic screens aimed at identifying small molecules that protect Arabidopsis seedlings from infection. One of the first chemicals identified through this screen was the sulfanilamide compound sulfamethoxazole (Smex). This compound was later shown to also reduce the susceptibility of both Arabidopsis and wheat to infection by the fungal pathogen Fusarium graminearum, suggesting a broad spectrum of activity. More detailed investigations of Smex indicated that the protective activity of this compound did not derive from antimicrobial effects, and that this activity was not executed through common defence-related signalling pathways. The folate biosynthetic pathway enzyme dihydropteroate synthase is a known target of sulfanilamides, and it does appear to contribute to Smex-induced disease resistance, albeit in a folate-independent manner. In order to identify downstream mediators of Smex activity, I initiated two forward genetic screens intended to recover mutants with altered sensitivity to Smex in a seedling growth assay. Interestingly, while these screens yielded mutants with striking Smex sensitivity phenotypes, disease resistance phenotypes were not altered. Gene expression profiling of Arabidopsis tissues treated with Smex prior to bacterial inoculation suggested that this compound generally affects lipid signalling. Altogether, it is evident that Smex elicits a complex set of responses in Arabidopsis with apparently non-overlapping phenotypic outputs.

Texte remanié de : Dissertation : Philosophie : Universität des Saarlandes : 1985. / Bibliogr. p. 351-359. Notes bibliogr. Index.

Plant hormones act in a complex network where their pathways regulate and interact to control different mechanisms, such as development and stress responses. This crosstalk between hormones can be exploited by pathogens to suppress plant defense responses and thereby increase pathogen growth. Pseudomonas syringae pathogenicity is reliant on a Type III secretion system (TTSS) that acts as a specialized injection apparatus to deliver virulence proteins, known as type III effectors (TTEs), into the plant cell cytosol. In my work, I have screened hormone inducible promoter::GUS transgenic Arabidopsis thaliana lines against a P. syringae TTE library in order to identify TTEs involved in the perturbation of hormone signaling in planta. Through this screen I identified two P. syringae TTEs, HopAK1 and HopAL1, both belonging to the same bacterial strain P. syringae pv. maculicola ES4326. I found that HopAK1 can sensitize A. thaliana plants to auxin. On the other hand, HopAL1 activates auxin signaling. Monitoring of auxin signaling was done using transgenic DR5::GUS plants. Both TTEs render the plant susceptible to bacterial infection, highlighting a potential relationship between increased auxin signaling and virulence.

Plant hormones act in a complex network where their pathways regulate and interact to control different mechanisms, such as development and stress responses. This crosstalk between hormones can be exploited by pathogens to suppress plant defense responses and thereby increase pathogen growth. Pseudomonas syringae pathogenicity is reliant on a Type III secretion system (TTSS) that acts as a specialized injection apparatus to deliver virulence proteins, known as type III effectors (TTEs), into the plant cell cytosol. In my work, I have screened hormone inducible promoter::GUS transgenic Arabidopsis thaliana lines against a P. syringae TTE library in order to identify TTEs involved in the perturbation of hormone signaling in planta. Through this screen I identified two P. syringae TTEs, HopAK1 and HopAL1, both belonging to the same bacterial strain P. syringae pv. maculicola ES4326. I found that HopAK1 can sensitize A. thaliana plants to auxin. On the other hand, HopAL1 activates auxin signaling. Monitoring of auxin signaling was done using transgenic DR5::GUS plants. Both TTEs render the plant susceptible to bacterial infection, highlighting a potential relationship between increased auxin signaling and virulence.

Doctor of Philosophy / Plant Pathology / Frank F. White / Rice and wheat are two major crops that suffer losses from the diseases of bacterial blight and bacterial leaf streak, which are caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas translucens pv. undulosa (Xtu), respectively. Transcriptional-Activator Like (TAL) effectors, a special family of type III effector proteins from Xanthomonas, have been demonstrated as critical virulence factors that act by inducing corresponding susceptibility (S) genes in several disease complexes of plants. In this study, I analyzed the contributions of TAL effectors from Xoo and Xtu to virulence and in modulating host gene expression to enhance susceptibility. Specifically, the TalC effector from the African Xoo strain AXO1947 was identified as a critical virulence factor, which functions by promoting expression of the gene OsSWEET14 in rice. TalC is interchangeable with other major TAL effectors from Asian strains of Xoo on the basis of functional complementation. The TAL effector PthXo2 from the Asian Xoo strain JXO1 is a major virulence factor and contains 21.5 repeats in the central repetitive region that targets OsSWEET13 in indica rice varieties but not in japonica rice varieties. A one repeat deletion in the PthXo2 effector enabled effector specificity to switch from indica rice to japonica rice. TAL effector genes from a genomic analysis of the Xtu strain XT4699 and related strains were characterized with regards to their involvement in virulence and the modulation of host gene expression in the Chinese Spring wheat cultivar. The identification of TAL effectors with virulence contributions and their target S genes is important for understanding the virulence mechanisms of Xanthomonas bacteria and promises to provide new strategies for disease control.

Master of Science / Entomology / C. Michael Smith / The bird cherry oat aphid, Rhopalosiphum padi, and one of the viruses it vectors, barley yellow dwarf virus (BYDV), form a very damaging pest unit on cereals. Understanding how people can better manage crops to prevent damage or recognize environmental or geographic factors that put their crops at risk for BYDV could lead to improved virus aphid management strategies. One of the most successful methods for mitigating pest damage is using pest-resistant varieties of crops. Seven candidate wheat varieties were screened for resistance to R. padi, by testing aphid population densities and aphid host choice. Results of this research identified six varieties of wheat that show resistance to R. padi. One of the major knowledge gaps in BYDV management is forecasting potential damage. Our objective was to create viral presence maps, to start building the foundation of correlations between persistent and changing frequency of viruliferous R. padi. The results of BYDV assays in R. padi in wheat fields across Kansas indicated that the amount of BYDV infection in viruliferous aphids changes rapidly from year to year, and differs considerably between geographic regions and field landscape characteristics in Kansas. Neonicotinoid wheat seed treatment is a management technique that reduces R. padi populations. However, a common question among producers is whether or not seed treatments stop viral transmission. Results of greenhouse seed treatment experiments with plants from neonicotinoid treated and untreated seed infested with viruliferous R. padi indicated no significant difference in viral transmission due to seed treatment. This new information about R. padi wheat varietal resistance, geographic distribution of BYDV in Kansas, and neonicotinoid seed treatment creates a better understanding of aphid-virus-wheat interactions. Results from this thesis directly enhance producer ability to forecast risk from BYDV, select wheat varieties that effectively reduce R. padi as a BYDV vector, and to make better decisions about the use of insecticide to reduce BYDV infection.

Doctor of Philosophy / Department of Plant Pathology / Xiaoyan Tang / P. syringae is a group of bacterial phytopathogens that can infect a wide variety of plants. These bacteria rely on the type III secretion system (TTSS) to deliver effectors into plant cells for infection. The TTSS genes, that encode the TTSS apparatus and the effectors, are repressed when bacteria grow in nutrient rich media but are strongly induced in the plants and in minimal medium (MM). Plant cutin monomers appear to negatively regulate the P. syringae TTSS genes. It is poorly understood how bacteria sense the environmental signals to regulate the TTSS genes. By genetic screen, four sets of transposon insertion mutants displaying aberrant TTSS gene expression were isolated: KB and fin mutants derepress the TTSS genes in rich medium KB and in the presence of a cutin monomer precursor in MM, respectively; min and pin mutants are defective in induction of TTSS genes in MM and in plants, respectively. A putative two-component sensor histidine kinase, RohS, is identified to be required for the induction of avrPto-LUC in MM and in plants. The rohS gene is in an operon containing a two-component response regulator gene rohR. Mutation of rohS in P. s. phaseolicola and P. s. tomato reduced the bacterial pathogenicity on hosts and HR-inducing activity on non-hosts. Our results suggested that RohS acts upstream of HrpR/HrpS. The phosphorylated RohR represses TTSS genes. It is likely that RohS acts as phosphatase of RohR in the TTSS-inducing conditions, and subsequently derepresses TTSS genes. Simple sugars such as glucose, sucrose and fructose are known to be inducers of the TTSS genes. Isolation of four min mutants defective in fructose-uptake enabled us to study if sugars serve as extracellular signals or as essential nutrients. Our results suggest that fructose acts as an essential nutrient for the activation of type III genes. These mutants slightly compromised induction of avrPto promoter in Arabidopsis and pathogenicity on the host bean plant, but displayed normal HR elicitation on non-host plant tobacco. The reduced pathogenicity suggested that exploitation of fructose from the host tissue is an important means for pathogenesis of P. s. phaseolicola.

Doctor of Philosophy / Department of Plant Pathology / Scot H. Hulbert / The rice cultivar Oryzica Llanos 5 (OL5) possesses a high level of resistance to the fungus Magnaporthe grisea. The number and chromosomal location of quantitative trait loci (QTL) conferring resistance against eight isolates of the blast fungus were tested in two different populations of recombinant inbred lines from the cross Fanny x OL5. Twenty one QTL were detected and associated with the resistance traits, disease leaf area and lesion type, on 9 rice chromosomes. Eight of these 21 resistance loci had significant resistance effects in both experiments, while the others had effects that were only statistically significant in one experiment. Most, but not all, of the QTL occurred in the same genomic regions as either genes with major race-specific effects or other resistance QTL that had been described in previous experiments. Most of the QTL appeared to be race-specific in their effects but it is possible some of the QTL with smaller effects were nonspecific. One of the blast isolates used was FL440, which causes limited disease on OL5 and was probably virulent on most or all of the major genes from OL5. Three QTL affected resistance to FL440 in both experiments, one of which mapped to a region on chromosome 9 where no blast resistance genes have yet been mapped. An advanced backcross strategy with marker-assisted selection for OL5 alleles in QTL regions was used to generate five BC2F3 populations carrying five different target regions associated with partial resistance to rice blast disease. Three of five of these populations were analyzed for segregation for resistance to the M. grisea isolate FL440. One QTL designated qrbr-11.3 near the bottom of rice chromosome 11 was found to be significantly associated with partial blast resistance in 120 lines of a BC2F3 population (P< 0.01). This QTL accounted for 12.4% and 8.0% of the phenotypic variation in diseased leaf area and lesion type observed under greenhouse inoculation. Examination of the genomic sequence at the qrbr-11.3 locus showed that twenty-nine candidate resistance genes are present at that locus (~1.8 Mb), twenty-seven of which are predicted NBS-LRR genes. Ultimately, the information from this study can be integrated into the development of improved lines with OL5-derived QTL for resistance.