Targeted gene mutation in Phytophthora spp.
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 necrosisinducing 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.
Lamour
Kurt H
KH
Department of Entomology and Plant Pathology, The University of Tennessee, Rm 205 Ellington Plant Science, 2431 Joe Johnson Dr., Knoxville 37996, USA. klamour@utk.edu
Finley
Ledare
L
Hurtado-Gonzales
Oscar
O
Gobena
Daniel
D
Tierney
Melinda
M
Meijer
Harold J G
HJ
D020418:Algal Proteins / Q000235:genetics*; D000595:Amino Acid Sequence; D005818:Genetic Engineering / Q000379:methods; D015698:Genomic Library; D005838:Genotype; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010641:Phenotype; D010838:Phytophthora / Q000235:genetics* / Q000254:growth & development; D016415:Sequence Alignment
D020418:Algal Proteins