Progress 01/01/05 to 12/31/05
Outputs
The goal of this project is to identify new sources of resistance (R) genes in plants effective against plant viruses. In this project, we have created a hybrid (designated N. edwardsonii var. Columbia) that could be used for the genetic characterization of new virus R genes. The feature that makes N. edwardsonii valuable is that it is a hybrid between a Nicotiana species that is susceptible to a large number of viruses (N. clevelandii) and a Nicotiana species that is resistant to those same viruses (N. glutinosa). We have identified at least 70 plant viruses, divided amongst 20 virus genera, that infect N. clevelandii, but cannot infect N. glutinosa. We have made crosses between N. edwardsonii var. Columbia and N. clevelandii to characterize a single dominant gene that specifies resistance to Tomato bushy stunt virus (TBSV), and we subsequently found that this locus also conditioned resistance to other tombusviruses, in particular Lettuce necrotic stunt virus,
Cymbidium ringspot virus, Cucumber necrosis virus and Carnation Italian ringspot virus. We have also developed a gene silencing assay that specifically targets host resistance genes that fall into the NBS-LRR category. The assay has initially been developed to target the N gene, a gene in N. edwardsonii that specifies resistance to Tobacco mosaic virus. We created a construct derived from exon 1 of the N gene that would result in a hairpin mRNA, which in turn would trigger posttranscriptional gene silencing. Transgenic plants that expressed the hairpin mRNA exhibited a 24 h to 72 h delay in HR development, and RT-PCR confirmed that the N gene transcript level was reduced in these plants. We also evaluated the plant response to TMV-30B-GFP, a TMV vector that expresses GFP. The N-silenced transgenic plants developed GFP-fluorescent lesions that could be visualized under UV fluorescence, whereas the N. edwardsonii controls developed necrotic lesions that did not flouresce. The assay with
TMV-30B-GFP confirmed that TMV was replicating in the N-silenced plants. We also developed a second set of transgenic plants in which we utilized a hairpin transgene that differed from the N gene by approximately 10%. These plants also exhibited a delayed HR phenotype in response to TMV inoculation. This study demonstrated that we could develop transgenic plants in which a block of R genes related by sequence could be shut off. We have now extended our gene silencing assay by showing that silencing of the N family of resistance genes abolishes resistance to TBSV. N-silenced transgenic plants consistently responded with a delayed HR, whereas the N. edwardsonii control had developed a strong local lesion response. We know that the N gene itself does not condition resistance to TBSV, as we had shown that TBSV anf TMV resistance segregated independently. Our hypothesis now is that we have silenced the entire family of N-related sequences and one of these N-related sequences encodes a TBSV
resistance gene. We have now created an N. glutinosa BAC library that consists of 37,000 clones with an average insert size of 120 kbp and are now identifying N-related resistance genes in the library.
Impacts
This project is important because it provides new, fundamental information about how to identify and clone new sources of virus resistance in plants. Tombusviruses such as TBSV and Lettuce necrotic stunt cause economically important diseases in a variety of crops such as lettuce and tomato. The resistance genes we identify will ultimately be useful for developing new strategies for resistance to pathogen attack. for example, our TBSV R gene could be moved into tomato varieties to protect them from TBSV, as tomatoes currently have no resistance to this virus. Furthermore, this study will provide new information about the structure of virus resistance genes.
Publications
- Cawly, J., Cole, A. B., Kiraly, L., Qiu, W., and Schoelz, J. E. 2005. The plant gene CCD1 selectively blocks cell death during the hypersensitive response to Cauliflower mosaic virus infection. Mol. Plant-Microbe. Interact. 18, 212-219.
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Progress 09/01/03 to 08/31/05
Outputs
OUTPUTS: The central tenet of the gene-for-gene theory is that a plant resistance (R) gene product recognizes a specific pathogen avirulence (Avr) gene product, which sets in motion a cascade of plant defenses. In many instances, the outcome of this interaction is a hypersensitive reaction (HR), a plant defense response that is characterized by a rapid death of a limited number of cells in the region of the invading pathogen. Of over 200 virus resistance genes that have been identified in crop plants, wild relatives, and model plant species such as Arabidopsis thaliana, 80% are inherited monogenically and many of these condition HR. Although a large number of virus resistance genes have been characterized genetically, only a handful have been physically cloned and their structure elucidated. In this study, we have sought to discover new R genes by exploring the nontarget effects associated with silencing of the N gene in Nicotiana edwardsonii, an amphidiploid species derived from N.
glutinosa and N. clevelandii. The N protein confers resistance to tobacco mosaic virus (TMV), and is representative of a family of NB-LRR proteins present in Nicotiana glutinosa. Previous studies have shown that silencing of the N gene or of other plant genes associated with N-mediated defenses abolishes host resistance to TMV, and this effect can be measured through enhancements in movement or replication of TMV in the N-silenced plants. However, the nontarget effects of gene silencing have not been investigated thoroughly. Notably, are the functions of other R genes also affected in experiments designed to silence the N gene? To investigate whether heterologous sequences could silence the N gene, we selected a resistance gene homolog from N. glutinosa that differed from the N gene by approximately 17%, created a hairpin transgene, and developed transgenic N. edwardsonii plants. Expression of this hairpin in the transgenic N. edwardsonii plants compromised the hypersensitive response
to TMV, demonstrating that a single hairpin transgene could silence a block of R genes related by sequence similarity. We then investigated whether the response of N-silenced plants to other viruses would be altered, and found that the hypersensitive response triggered against the tombusviruses, tomato bushy stunt virus and cymbidium ringspot virus, was also compromised. This study indicates that a tombusvirus R gene shares some homology to the N gene, which could facilitate the cloning of this gene.
PARTICIPANTS: Individuals who worked on this project include a postdoctoral associate, Dr. Boovharaghan Balaji, and three graduate students: John Cawly, Anthony Cole, and Carlos Angel. Both John Cawly and Anthony cole completed their PhDs while working on this project, whereas Dr. Balaji completed his postdoctoral training and obtained a job with Dupont in India. Carlos Angel remains a graduate student in my lab. I have also formeed a collaboration with Lorant Kiraly at the Plant Protection Institue in budapest, Hungary.
TARGET AUDIENCES: This information was disseminated to individuals that attended the Amercian Society for Virology meetings and the American Phytopathological Society meetings. In addition, aspects of this research has been incorporated into an undergraduate course in introductory plant pathology and a graduate course in plant virology.
Impacts
In this study, we showed that strategies designed to target the N gene for post-transcriptional gene silencing also target a family of N gene homologs present in Nicotiana species. As a prerequisite to the silencing study, we used a PCR-based approach to estimate the number of N gene homologs in Nicotiana species that might be affected by silencing of the N gene. We found that N. glutinosa contained at least 10 homologs in addition to the N gene, whereas N. clevelandii contained at least 4 homologs. Once we confirmed that the N gene in N. edwardsonii had been silenced, we then investigated whether the response of N-silenced plants to viruses other than TMV would be altered. We found that the HR triggered against the tombusviruses TBSV and CymRSV was also compromised. Our results, therefore, suggest that the nucleotide sequence of the tombusvirus resistance gene must have a high degree of homology to the N gene. Our observation that silencing of the N gene affects
resistance to both TBSV and CymRSV indicates that their resistance genes are related at least at the sequence level, and this information should facilitate the cloning of these new R genes. Our demonstration that gene silencing technology can be effective in identifying a previously unrecognized tombusvirus R gene has the potential to augment existing techniques for characterization and cloning new R genes. In particular, it could facilitate the cloning of valuable disease resistance genes in plants such as N. edwardsonii, a species that could serve as a reservoir for virus R genes but for which no physical map exists. It is impractical to isolate genomic clones of R genes and randomly test them individually for their reaction to a specific pathogen, but it is nevertheless possible to find new R gene specificities using a gene silencing assay that shuts off entire blocks of related R genes. This assay could narrow the search to a manageable number of R gene candidates that could be
identified through southern hybridization of an N. glutinosa genomic library. Previous work has shown that the TBSV P22 protein is responsible for elicitation of HR in N. edwardsonii. Consequently, individual R genes could then be tested for HR activity through co-agroinfiltration with the TBSV p22 gene into a susceptible solanaceous host such as N. clevelandii.
Publications
- Balaji, B., Cawly, J., Angel., C., Zhang, Z., Palanichelvam, K., Cole, A., and Schoelz, J. 2007. Silencing of the N family of resistance genes in Nicotiana edwardsonii compromises the hypersensitive response to tombusviruses. Mol Plant-Microbe-Interact. 20, 1262-1270.
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Progress 01/01/04 to 12/31/04
Outputs
The goal of this project is to provide a greater understanding of how plant viruses cause disease and how plants resist infection by plant viruses. In this project, we have characterized a new variety of Nicotiana edwardsonii (designated N. edwardsonii var. Columbia) that we have synthesized which could be used for the genetic characterization of as many as 70 viral R genes. The feature that makes N. edwardsonii valuable is that it is a hybrid between a Nicotiana species that is susceptible to a large number of viruses (N. clevelandii) and a Nicotiana species that is resistant to those same viruses (N. glutinosa). By cross referencing information in published reports and from the VIDE virus database, we have identified at least 70 plant viruses, divided amongst 20 virus genera, that can infect N. clevelandii, but cannot infect N. glutinosa. The original N. edwardsonii could not be crossed successfully with either parent, because the hybrids were sterile. However, we
found that the hybrid we synthesized differed in many aspects from the original N. edwardsonii, but most importantly, the progeny of crosses with either N. glutinosa or N. clevelandii were fertile. To distinguish our hybrid from the original N. edwardsonii, we designated our plant, N. edwardsonii var. Columbia. We have made crosses between N. edwardsonii var. Columbia and N. clevelandii to characterize a single dominant gene that specifies resistance to Tomato bushy stunt virus (TBSV), and we subsequently found that this locus also conditioned resistance to other tombusviruses such as Lettuce necrotic stunt virus, Cymbidium ringspot virus, Cucumber necrosis virus and Carnation Italian ringspot virus. We have also developed a gene silencing assay that specifically targets host resistance genes that fall into the NBS-LRR category. The assay has initially been developed to target the N gene, a gene in N. edwardsonii that specifies resistance to Tobacco mosaic virus. We created a
construct derived from exon 1 of the N gene that would result in a hairpin mRNA, which in turn would trigger posttranscriptional gene silencing. Transgenic plants that expressed the hairpin mRNA exhibited a 24 h to 72 h delay in HR development, and RT-PCR confirmed that the N gene transcript level was reduced in these plants. We have now extended our gene silencing assay by showing that the transgenic plants also exhibit a delayed HR in response to TBSV. In one experiment, we inoculated one half of the leaf from an N gene-silenced N. edwardsonii plant with TMV and the other half with TBSV. At three dpi, the TMV-inoculated half had not developed lesions, whereas the N. edwardsonii control exhibited well-developed lesions. Similarly, the TBSV-inoculated half leaf had not developed lesions, whereas the TBSV-inoculated N. edwardsonii control had developed a strong local lesion response. Our hypothesis now is that one of these N-related sequences encodes a TBSV resistance gene.
Furthermore, these results are consistent with our hypothesis that the N gene is representative of a family of NB-LRR genes in Nicotiana, and that silencing of the N gene would silence other family members as well.
Impacts
This project is important because it provides new, fundamental information about how viral pathogens cause disease in plants. This information will ultimately be useful for developing new strategies for resistance to pathogen attack.
Publications
- Cole, A.B., Kiraly, L., Lane, L.C., B.E. Wiggins, Ross, K., and Schoelz, J.E. 2004. Temporal expression of PR-1 and enhanced mature plant resistance to virus infection is
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Progress 01/01/03 to 12/31/03
Outputs
This goal of this project is to provide a greater understanding of how plant viruses cause disease and how plants resist infection by plant viruses. In this project, we have characterized a new variety of Nicotiana edwardsonii (designated N. edwardsonii var. Columbia) that we have synthesized which could be used for the genetic characterization of as many as 70 viral R genes. The feature that makes N. edwardsonii valuable is that it is a hybrid between a Nicotiana species that is susceptible to a large number of viruses (N. clevelandii) and a Nicotiana species that is resistant to those same viruses (N. glutinosa). By cross referencing information in published reports and from the VIDE virus database, we have identified at least 70 plant viruses, divided amongst 20 virus genera, that can infect N. clevelandii, but cannot infect N. glutinosa. The original N. edwardsonii could not be crossed successfully with either parent, because the hybrids were sterile. However, we
found that the hybrid we synthesized differed in many aspects from the original N. edwardsonii, but most importantly, the progeny of crosses with either N. glutinosa or N. clevelandii were fertile. To distinguish our hybrid from the original N. edwardsonii, we designated our plant, N. edwardsonii var. Columbia. To determine how virus resistance genes would segregate in interspecific crosses between Columbia and N. clevelandii, we followed the fate of the N gene, a single dominant gene that specifies resistance to Tobacco mosaic virus (TMV). We found that the entire chromosome that contains the N gene was introgressed into N. clevelandii to create an addition line. The putative N. clevelandii addition line was homozygout for the N gene and contained 50 chromosomes instead of the 48 characteristic of N. clevelandii. These plants are being evaluated to identify other virus resistance genes that might reside on the N gene chromosome. To date, we have found that resistance to Cauliflower
mosaic virus (CaMV) and Tomato bushy stunt virus (TBSV) must reside on other chromosomes. In another series of crosses, we are characterizing the inheritance of resistance to CaMV and TBSV. Thus, we can use N. edwardsonii var. Columbia as a bridge to move resistance genes from N. glutinosa to N. clevelandii and characterize their inheritance. To develop molecular evidence that an N. glutinosa chromosome was introgressed into N. clevelandii, we utilized a PCR-based approach to adapt markers from the tomato genome map. Enough synteny exists between Nicotiana and tomato such that primer sequences developed against the tomato genome genome may be used for the creation of Nicotiana markers. These markers could be used to anchor the N gene and other virus resistance genes to the tomato genome map.
Impacts
This project is important because it provides new, fundamental information about how viral pathogens cause disease in plants. This information will ultimately be useful for developing new strategies for resistance to pathogen attack.
Publications
- Schoelz, J.E., Palanichelvam, K., Cole, A.B., Kiraly, L., and Cawly, J. 2003. Dissecting the avirulence and resistance components that comprise the hypersensitive response to Cauliflower mosaic virus in Nicotiana. In "Plant-Microbe Interactions, Vol. 6" (Gary Stacey and Noel Keen, eds) The American Phytopathological Society pp 259-284.
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