|White, David - Washington State University|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 4/28/2009
Publication Date: 6/21/2009
Citation: White, D., Chen, W. 2009. Identifying pathogenic determinants of Ascochyta rabiei via genetic complementation. Meeting Abstract.
Technical Abstract: Introduction – The necrotrophic pathogen Ascochyta rabiei causes chickpea ascochyta blight, an economically important disease worldwide. Despite extensive investigations into the biology and epidemiology of the disease, very little is known about the molecular mechanisms of the pathogen. The objective of this research is to identify pathogenicity determinants of A. rabiei using complementation tests. Materials and Methods –The mutant strain ArW519 was non-pathogenic on chickpea generated from wild-type strain AR628 as a result of a single T-DNA insertion event. Genomic DNA flanking the T-DNA insertion was used as a probe to isolate genomic DNA clones from a phage library of the strain AR628 genome. These genomic fragments were moved separately into a T-DNA shuttle vector with geneticin resistance and re-transformed into the ArW519 genome. The T-DNA insertion was verified by resistance to both hygromycin and geneticin and by PCR. Both double recombination events, resulting in replacement of the original T-DNA with new T-DNA as well as novel integration sites of the new T-DNA were recovered. The pathogenicity of eight complemented ArW519 mutants recovered from independent T-DNA integration events were compared to that of the parent ArW519 mutant and of wild-type AR628 on chickpea cultivars Spanish White and Dwelley using a minidome bioassay. Results and Discussion – Six clones were isolated from the AR628 library using the ArW519 probe, ranging from 4016 bp to 5529 bp. Each clone was independently re-introduced into the ArW519 genome and tested for the restoration of pathogenicity. Only one clone was able to functionally restore pathogenicity to the ArW519 mutant. For the re-integration of each region, the identical T-DNA molecule, conferring resistance to geneticin, was utilized. For each re-integration, nearly three quarters of the recovered geneticin-resistant A. rabiei transformants were no longer resistant to hygromycin, suggesting that the newly introduced T-DNA region had simply replaced the original T-DNA integrant. DNA isolated from transformants that were resistant to both hygromycin and geneticin was used as template for primers specific for each of the antibiotic cassettes to verify that both genes were intact. The genomic fragment that restores pathogenicity to the ArW519 mutant contains about 3000 bp of DNA upstream of the T-DNA insertion and about 1000 bp of DNA downstream. When compared to sequence databases this A. rabiei genomic fragment carries regions that are similar to retrotransposon Molly from Stagonospora nodorum and the AvrLM1 avirulence gene from Leptosphaeria maculans. The ability to successfully complement non-pathogenic A. rabiei mutants is an important step to better understand the underlying pathogenic mechanisms of the pathogen.