Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various "reverse genetic" approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants.
A genetic screen of a population of Arabidopsis thaliana lines exhibiting enhanced somatic homologous recombination yielded a mutant affected in expression of a gene encoding a caltractin-like protein (centrin). The hyperrecombinogenic phenotype could be reproduced using RNA interference (RNAi) technology. Both the original mutant and the RNAi plants exhibited a moderate UV-C sensitivity as well as a reduced efficiency of in vitro repair of UV-damaged DNA. Transcription profiling of the mutant showed that expression of components of the nucleotide excision repair (NER) pathway and of factors involved in other DNA repair processes were significantly changed. Our data suggest an indirect involvement of centrin in recombinational DNA repair via the modulation of the NER pathway. These findings thus point to a novel interconnection between an early step of NER and homologous recombination, which may play a critical role in plant DNA repair.
The transferred DNA (T-DNA) is transported from Agrobacterium tumefaciens to the nucleus and is stably integrated into the genome of many plant species. It has been proposed that the VirD2 protein, tightly attached to the T-DNA, pilots the T-DNA into the plant cell nucleus and that it is involved in integration. Using agroinfection and beta-glucuronidase expression as two different very sensitive transient assays for T-DNA transfer, together with assays for stable integration, we have shown that the C-terminal half of the VirD2 protein and the VirD3 protein are not involved in T-DNA integration. However, the bipartite nuclear localization signal, which is located within the C terminus of the VirD2 protein and which has previously been shown to be able to target a foreign protein into the plant cell nucleus, was shown to be required for efficient T-DNA transfer. virD4 mutants were shown by agroinfection to be completely inactive in T-DNA transfer.
Agrobacterium tumefaciens is routinely used to engineer desirable genes into dicotyledonous plants. However, the economically important graminaceous plant maize is refractory to tumor induction by inoculation with virulent strains ofA. tumefaciens. Currently, the only clearcut evidence for transferred DNA (T-DNA) transport fromAgrobacterium to maize comes from agroinfection. To study T-DNA transfer from Agrobacterium to maize cells in a virus-free system, we used here the fi-glucuronidase (GUS; EC 3.2
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.