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Targeted Gene Mutation in Phytophthora spp.

December 2006 , Volume 19 , Number  12
Pages  1,359 - 1,367

Kurt H. Lamour , 1 Ledare Finley , 1 Oscar Hurtado-Gonzales , 1 Daniel Gobena , 1 , 2 Melinda Tierney , 3 and Harold J. G. Meijer 2

1The University of Tennessee, Department of Entomology and Plant Pathology, Rm 205 Ellington Plant Science, 2431 Joe Johnson Dr., Knoxville 37996, U.S.A.; 2Laboratory of Phytopathology, Plant Sciences Group, Wageningen University, Binnenhaven 5, NL-6709 PD Wageningen, The Netherlands; 3The University of Tennessee and Oak Ridge National Laboratory, Genome Science and Technology Graduate Program, 1060 Commerce Park, Oak Ridge 37830-8026, U.S.A.

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Accepted 12 July 2006.

The genus Phytophthora belongs to the oomycetes and is composed of plant pathogens. Currently, there are no strategies to mutate specific genes for members of this genus. Whole genome sequences are available or being prepared for Phytophthora sojae, P. ramorum, P. infestans, and P. capsici and the development of molecular biological techniques for functional genomics is encouraged. This article describes the adaptation of the reverse-genetic strategy of targeting induced local lesions in genomes (TILLING) to isolate gene-specific mutants in Phytophthora spp. A genomic library of 2,400 ethylnitrosourea (ENU) mutants of P. sojae was created and screened for induced point mutations in the genes encoding a necrosis-inducing protein (PsojNIP) and a Phytophthora-specific phospholipase D (PsPXTM-PLD). Mutations were detected in single individuals and included silent, missense, and nonsense changes. Homozygous mutant isolates carrying a potentially deleterious missense mutation in PsojNIP and a premature stop codon in PsPXTM-PLD were identified. No phenotypic effect has yet been found for the homozygous mutant of PsojNIP. For those of PsPXTM-PLD, a reduction in growth rate and an appressed mycelial growth was observed. This demonstrates the feasibility of target-selected gene disruption for Phytophthora spp. and adds an important tool for functional genomic investigation.

Additional keywords: knockout , SNP .

© 2006 The American Phytopathological Society