No description available.

The topic of this thesis is focused on the testing of resistance of selected Brassica species to the black rot infection and viral mosaics caused by economically important pathogens of Brassicaceae family. The theoretical part describes characteristics of causal pathogens - Xanthomonas campestris pv. campestris (Xcc), Turnip mosaic virus (TuMV) and Turnip yellow mosaic virus (TYMV), and summarize the current state of a resistance study of these pathogens in the Brassicaceae family. The thesis also describes modern molecular methods used for the detection of bacterial and viral pathogens. In the experimental part, the detections of Xcc, TuMV and TYMV pathogens were optimized by PCR and RT-PCR. For bacterium Xcc, the Real-time PCR targeting a part of the zur gene sequence was designed using a TaqMan® probe. This detection system was subsequently processed in the form of a certified methodology for use in diagnostics. To increase the specificity, Real-time PCR targeting zur gene was involved in the Multiplex Real time PCR reaction. Then the dynamics of the Xcc infection was monitored in 6 hybrid cabbage cultivars. The testing of resistance to the black rot disease was optimized by the procedure including artificial inoculations using the suspension of the Xcc isolates HRIW 3811, 3971A and 1279A and the SU1 isolate originated from the Czech Republic. In a four-year experiment, the total of 42 homozygous breeding lines and 4 hybrid cultivars were tested, where 5 lines were recommended for breeding for resistance to the black rot disease. For the detection of TuMV and TYMV viruses, Real-time RT-PCR approaches based on the TaqMan® probe and SYBR Green dye were tested. The target region of both detections was the coat protein. The TuMV detection has been optimized for SYBR Green approach; for the TYMV detection, the use of the TaqMan® probe has been recommended. Detection systems were used to evaluate artificial inoculations of 6 cabbage cultivars by individual viruses. The tested plants did not show visual symptoms of infection therefore the presence of viruses was evaluated by Real-time RT PCR. The system designed for TYMV detected the presence of virus in all tested samples, TuMV was detected only in two samples. Negative detection results are probably in connection with the absence of TuMV symptoms which indicates unsuccesful plant inoculation. For both detection systems, it was recommended the verification on a wider range of viral isolates prior to standard use in diagnostics

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 1977 / Vita. / Includes bibliographical references. / Ph. D. / Ph. D. Massachusetts Institute of Technology, Department of Biology

TCV (Turnip crinkle virus) coat protein is required for the resistance response in Arabidopsis thaliana Di-17 plants. An aspartate to asparagine mutation at amino acid four of the coat protein is sufficient to result in resistance-breaking. To determine the essential chemical properties responsible for the induction of resistance, a series of site-directed mutants were produced. Serine as well as asparagine at amino acid four induces systemic disease on both Di-3 and Di-17 plants; however, replacement of aspartate with glutamate retains the ability to induce the HR (hypersensitive response) and resist TCV infection with rapid and strong induction of PR-1 gene. These data suggest that the negative charge at the fourth amino acid of the coat protein is critical for the induction of resistance. Taken together with other mutagenesis research, the N-terminus of the coat protein appears to be the sole viral recognition element. The A. thaliana TIP protein is suggested to be involved in resistance, mainly through its C-terminus. Interestingly, one of the resistance-breaking mutants (D4N) produces a HR on Di-3 plants that are normally susceptible. The Di-3 TIP protein has several differences from the Di-17 TIP. To detect whether the delayed HR is related to interaction between Di-3 TIP and D4N mutation, a yeast two-hybrid assay was attempted. Interactions have not yet been detected. There are a number of possible explanations.

Infection of the resistant Arabidopsis ecotype Di-17 with Turnip Crinkle Virus (TCV) elicits hypersensitive response (HR), accompanied by increased expression of defense genes. HR to TCV is conferred by HRT, which encodes a coiled-coil (CC)-nucleotide-binding site (NBS)-leucine-rich repeat (LRR) class of resistance (R) protein. In contrast to HR, resistance requires HRT and a recessive locus designated rrt. Unlike most CC-NBS-LRR R proteins, HRT-mediated resistance is dependent on EDS1 and independent of NDR1. Resistance is also dependent on salicylic acid (SA) pathway and light. A dark treatment, immediately following TCV inoculation, suppresses HR, resistance and activation of a majority of the TCV-induced genes. To determine the genetic, molecular and biochemical basis of light-dependent defense pathway, we studied the role of various photoreceptors in HRT-mediated resistance to TCV, HRT protein levels and its localization. Interestingly, mutation in blue-light photoreceptors led to degradation of HRT via a proteasome-dependent pathway and resulted in susceptibility to TCV. Exogenous application of SA induced transcription of HRT, which restored HRT levels in some, but not all, mutant backgrounds. These results show that different photoreceptors function distinctly in maintaining post-transcriptional stability of HRT. In addition to photoreceptors, HRT also forms a complex with several other proteins, many of which participate in the RNA silencing pathway and are required for HRT-mediated resistance. Together, our results suggest that HRT forms a multi-protein complex and that HRT-mediated signaling involves reconstitution of this complex.

No description available.

No description available.

An interaction was recently detected between the potyviral protein, genome-linked (VPg) and the Arabidopsis thaliana translation initiation factor eIF(iso)4E (Wittmann et al., 1997). / Here, experiments were undertaken to address biological aspects of the VPg-eIF4E interaction. First, coimmunoprecipitation experiments performed with purified recombinant proteins have shown that VPg not only associates with eIF4E, as was previously published, but also with the larger eIF4F complex, of which eIF4E is a subunit. These results were confirmed by ELISA-type binding assays. It was also shown that there is no direct interaction between VPg and the other subunit of eIF4F, namely eIF4G. Finally, with the same experimental system, it was shown that the presence of eIF4G does not influence the binding affinity of VPg and eIF4E. / The interaction of VPg with the plant translation apparatus suggests that potyviral infection may alter the host protein expression profile. This hypothesis was investigated with the use of a protoplast system. We have shown that the global rates of protein synthesis in protoplasts transfected with an infectious TuMV cDNA clone dropped shortly after transfection, by as much as an estimated 70%. Recovery to normal levels occurred within 48 hours. / Evidence was obtained that the interaction between VPg and eIF4E is instrumental in this transient down-regulation of protein expression: protoplasts transfected with a mutant TuMV cDNA clone, the VPg of which has no affinity for eIF4E, failed to exhibit the drop in protein synthesis observed with the wild-type clone.

Turnip mosaic potyvirus (TuMV) is a single-stranded RNA plant virus. One of its proteins, the cylindrical inclusion (CI) protein, was hypothesized to interfere with host transcription via interaction with histone H3 protein. Interaction between CI and histone H3 was previously observed in Dr. Fortin's laboratory. Based on previous studies that demonstrated the importance of the H3 tail domain in gene regulation and chromosome arrangement, it was hypothesized that CI would interact with the tail rather than the globular domain. The objective of this project was to identify which histone H3 domains CI protein interacts with. The full-length, globular, and tail domains of histone H3 DNA were expressed in E. coli and purified. Based on in vitro interaction experiments, the CI protein was observed to interact with the globular domain of histone H3.

Turnip mosaic virus (TuMV) infects a variety of crops, worldwide, including the economically relevant Brassicacea family. It was previously demonstrated that TuMV infection in tobacco protoplasts leads to an overall decrease of host protein. However, it remains unclear whether this phenomenon is due to the repression of plant gene transcription during the infection period or due to viral inhibition of host translation. In this study, quantification of various transcripts and protein products from infected tobacco was performed via real-time RT-PCR and ELISA. In comparison to the gamma-tubulin endogenous control, gene expression for the tobacco H3, HSP70 and granule-bound starch synthase was affected by TuMV infection with time. / Tobacco protein accumulation in whole leaf tissues was also significantly affected by increase of virus particles.