Recipient Organization
UNIVERSITY OF VERMONT
(N/A)
BURLINGTON,VT 05405
Performing Department
Plant Biology
Non Technical Summary
Oomycetes are responsible for many important plant diseases, including potato late blight that devastated Ireland 160 years ago and is reemerging today, and sudden oak death that was recognized in 1995 and is causing large diebacks of western USA oak species. This group also includes downy mildew pathogens, which are important diseases of many crops and fruit species. The project is aimed at understanding a component of the plant defense system that is defined by the NIP1 gene in Arabidopsis, which confers partial resistance against downy mildew. The research will provide more understanding of the function of NIP1, as well as provide a precise genetic map position for the gene, which will facilitate its future cloning and characterization. Ultimately, after the gene is cloned, we may be able to use the information gained from study of NIP1 to design novel strategies for control of oomycete plant pathogens.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The objectives of this project are to further characterize an Arabidopsis thaliana mutant (family Brassicaceae) called nip1, and to genetically map the locus to a 2 cM region on chromosome 3. Mutations in the NIP1 gene cause plants to have compromised resistance an oomycete pathogen Hyaloperonospora parasitica (Hp) recognized by resistance genes at the RPP1 locus, leading nip1 plants to display enhanced susceptibility to this Hp isolate. Characterization of nip1 will involve assessing whether the mutation affects transcription of the resistance genes at the RPP1 locus, or two known defense genes that mediate signaling from RPP1. Additional studies will assess which plant developmental stages are most affected by the nip1 mutations to gain insight to the role of NIP1 in defense. High-resolution genetic mapping will be performed by screening F2 populations derived from crosses of nip1 to mapping lines for heightened susceptibility to Hp, and analyzing molecular markers for linkage to the NIP1 locus. Cloning the NIP1 gene is not feasible during the one-year term of this project. The expected product of this work will be a manuscript describing the phenotype of nip1 plants, and knowledge of the precise location of NIP1 that will potentiate its future map-based cloning.
Project Methods
To assess gene expression levels of RPP1a, RPP1b, RPP1c, EDS1 and PAD4 loci, nip1 and wild type plants, before and after pathogen infection, will be used to prepare mRNA. Samples will be analyzed using reverse transcriptase-PCR, or real time PCR to quantify transcript levels of each gene, as well as constitutively expressed control genes. Disease susceptibility will be assessed on plants at the cotyledon and true leaf stages following inoculation with conidia from the downy mildew pathogen. To map NIP1, segregating nip1 plants from an F2 mapping population will be screened for the enhanced pathogen susceptibility phenotype consistent with homozygous nip1, and then analyzed using a variety of molecular markers, including single nucleotide polymorphisms (SNPs), and co-dominant amplified polymorphic markers (CAPS). Approximately 10,000 F2 plants will be examined for the nip1 mutant phenotype, screened by PCR to assess presence of RPP1, and subjected to mapping using SNP and CAPs markers.