Discovery of Pathogenicity Genes in the Crucifer Anthracnose Fungus Colletotrichum higginsianum, Using Random Insertional Mutagenesis
Agrobacterium tumefaciens–mediated transformation (ATMT) was used for random insertional mutagenesis to identify pathogenicity genes in the hemibiotrophic fungus Colletotrichum higginsianum. A high-throughput primary infection assay on Arabidopsis thaliana seedlings allowed the rapid screening of 8,...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
The American Phytopathological Society
2009-02-01
|
| Series: | Molecular Plant-Microbe Interactions |
| Online Access: | https://apsjournals.apsnet.org/doi/10.1094/MPMI-22-2-0143 |
| _version_ | 1811286305400684544 |
|---|---|
| author | Aurélie Huser Hiroyuki Takahara Wolfgang Schmalenbach Richard O'Connell |
| author_facet | Aurélie Huser Hiroyuki Takahara Wolfgang Schmalenbach Richard O'Connell |
| author_sort | Aurélie Huser |
| collection | DOAJ |
| description | Agrobacterium tumefaciens–mediated transformation (ATMT) was used for random insertional mutagenesis to identify pathogenicity genes in the hemibiotrophic fungus Colletotrichum higginsianum. A high-throughput primary infection assay on Arabidopsis thaliana seedlings allowed the rapid screening of 8,850 transformants. Forty mutants showing reproducible pathogenicity defects on Arabidopsis and Brassica plants were obtained, and their infection phenotypes were characterized microscopically. Six mutants were impaired in appressorial melanization, fifteen had reduced penetration ability, 14 induced host papillae or hypersensitive cell death, and five were affected in the transition from biotrophy to necrotrophy. Southern blot analysis showed 58% of the transformants had single-site T-DNA integrations. Right-border flanking sequences were recovered from 12 mutants by inverse polymerase chain reaction (PCR) or thermal asymmetric interlaced PCR and were used to isolate the tagged genes from a genomic library. The putative pathogenicity genes encoded homologs of a major facilitator superfamily phosphate transporter, importin-β2, ornithine decarboxylase, β-1,3(4)-glucanase, ATP-binding endoribonuclease, carbamoyl-phosphate synthetase, and the polyprotein precursor of N-acetylglutamate kinase and N-acetylglutamyl-phosphate reductase. Six further loci were homologous to proteins of unknown function. None of these genes were previously implicated in the pathogenicity of any Colletotrichum species. The results demonstrate that ATMT is an effective tool for gene discovery in this model pathogen. |
| first_indexed | 2024-04-13T02:57:42Z |
| format | Article |
| id | doaj.art-595ceae3047a4c3584bb1ca0c5f9a836 |
| institution | Directory Open Access Journal |
| issn | 0894-0282 1943-7706 |
| language | English |
| last_indexed | 2024-04-13T02:57:42Z |
| publishDate | 2009-02-01 |
| publisher | The American Phytopathological Society |
| record_format | Article |
| series | Molecular Plant-Microbe Interactions |
| spelling | doaj.art-595ceae3047a4c3584bb1ca0c5f9a8362022-12-22T03:05:34ZengThe American Phytopathological SocietyMolecular Plant-Microbe Interactions0894-02821943-77062009-02-0122214315610.1094/MPMI-22-2-0143Discovery of Pathogenicity Genes in the Crucifer Anthracnose Fungus Colletotrichum higginsianum, Using Random Insertional MutagenesisAurélie HuserHiroyuki TakaharaWolfgang SchmalenbachRichard O'ConnellAgrobacterium tumefaciens–mediated transformation (ATMT) was used for random insertional mutagenesis to identify pathogenicity genes in the hemibiotrophic fungus Colletotrichum higginsianum. A high-throughput primary infection assay on Arabidopsis thaliana seedlings allowed the rapid screening of 8,850 transformants. Forty mutants showing reproducible pathogenicity defects on Arabidopsis and Brassica plants were obtained, and their infection phenotypes were characterized microscopically. Six mutants were impaired in appressorial melanization, fifteen had reduced penetration ability, 14 induced host papillae or hypersensitive cell death, and five were affected in the transition from biotrophy to necrotrophy. Southern blot analysis showed 58% of the transformants had single-site T-DNA integrations. Right-border flanking sequences were recovered from 12 mutants by inverse polymerase chain reaction (PCR) or thermal asymmetric interlaced PCR and were used to isolate the tagged genes from a genomic library. The putative pathogenicity genes encoded homologs of a major facilitator superfamily phosphate transporter, importin-β2, ornithine decarboxylase, β-1,3(4)-glucanase, ATP-binding endoribonuclease, carbamoyl-phosphate synthetase, and the polyprotein precursor of N-acetylglutamate kinase and N-acetylglutamyl-phosphate reductase. Six further loci were homologous to proteins of unknown function. None of these genes were previously implicated in the pathogenicity of any Colletotrichum species. The results demonstrate that ATMT is an effective tool for gene discovery in this model pathogen.https://apsjournals.apsnet.org/doi/10.1094/MPMI-22-2-0143 |
| spellingShingle | Aurélie Huser Hiroyuki Takahara Wolfgang Schmalenbach Richard O'Connell Discovery of Pathogenicity Genes in the Crucifer Anthracnose Fungus Colletotrichum higginsianum, Using Random Insertional Mutagenesis Molecular Plant-Microbe Interactions |
| title | Discovery of Pathogenicity Genes in the Crucifer Anthracnose Fungus Colletotrichum higginsianum, Using Random Insertional Mutagenesis |
| title_full | Discovery of Pathogenicity Genes in the Crucifer Anthracnose Fungus Colletotrichum higginsianum, Using Random Insertional Mutagenesis |
| title_fullStr | Discovery of Pathogenicity Genes in the Crucifer Anthracnose Fungus Colletotrichum higginsianum, Using Random Insertional Mutagenesis |
| title_full_unstemmed | Discovery of Pathogenicity Genes in the Crucifer Anthracnose Fungus Colletotrichum higginsianum, Using Random Insertional Mutagenesis |
| title_short | Discovery of Pathogenicity Genes in the Crucifer Anthracnose Fungus Colletotrichum higginsianum, Using Random Insertional Mutagenesis |
| title_sort | discovery of pathogenicity genes in the crucifer anthracnose fungus colletotrichum higginsianum using random insertional mutagenesis |
| url | https://apsjournals.apsnet.org/doi/10.1094/MPMI-22-2-0143 |
| work_keys_str_mv | AT aureliehuser discoveryofpathogenicitygenesinthecruciferanthracnosefunguscolletotrichumhigginsianumusingrandominsertionalmutagenesis AT hiroyukitakahara discoveryofpathogenicitygenesinthecruciferanthracnosefunguscolletotrichumhigginsianumusingrandominsertionalmutagenesis AT wolfgangschmalenbach discoveryofpathogenicitygenesinthecruciferanthracnosefunguscolletotrichumhigginsianumusingrandominsertionalmutagenesis AT richardoconnell discoveryofpathogenicitygenesinthecruciferanthracnosefunguscolletotrichumhigginsianumusingrandominsertionalmutagenesis |