Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108
Performing Department
Plant Biology
Non Technical Summary
Anti-microtubule herbicides, also called mitotic disrupter herbicides, are important in control of annual weed grasses for a wide variety of crop plants and turf grass. Used as pre-emergent herbicides, the dinitroanilines Treflan and Surflan (trifluralin and oryzalin, respectively) result in root tip swelling and seedling death due to disruption of microtubule processes during cell division (Sherman et al., 1996). Although the dinitroaniline herbicides have been shown to bind to plant tubulin (Strachan and Hess, 1983; Murthy et al., 1994), the major protein component of microtubules, genetic studies in our laboratories have shown that mutations conferring resistance to these herbicides reside in a number of genes other than tubulin. We propose to determine the molecular basis for anti-microtubule herbicide resistance by isolating and characterizing genes affected by resistant mutations. This project fits well with the stated goals for use of Hatch/MAES funds to 1) "promote research on fundamental biological problems related to national and local goals for agriculture" and 2) "enhance long-term research support for Plant Biology faculty by supporting short-term projects that have the potential to attract competitive research funding from NIFA or other external sources". The project clearly meets aim 1 in that understanding microtubule assembly and its regulation are critically important to understanding how cell walls are laid down, how mitotic and meiotic spindles function and how plants develop resistance to anti-microtubule herbicides. For the second aim, this project fits into a related project in which we have recently discovered that resistance to a different class of anti-microtubule herbicides, the phosphoric amides such as amiprophos-methyl, is caused by mutations in the heat shock cognate genes HSP70 and HSP40. The combination of the experiments in this proposal and the completed work on phosphoric amide herbicides and HSP cognate genes will allow us to characterize a large network genes regulating microtubule assembly and function. This project fits well in NIFA Research Problem Area 206. Basic Plant Biology
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
1. Positional cloning of ORY1, COR1, and SUPCS1 genes. We will used a map-based strategy that has been successful in our labs for cloning several genes (Nguyen et al., 2005). The strategy relies on a polymorphic field isolate, S1D2, that is interfertile with the laboratory strain but has approximately 2.7 nucleotide substitutions and 0.54 indels per 100 bp of noncoding sequence when compared with the laboratory strain. We used this strain to prepare a physical map of the Chlamydomonas nuclear genome using SNP markers (Kathir et al., 2003). Abundant simple sequence repeats (SSR) and transposable elements are listed on the Joint Genomes Institute genome sequence database and provide another source for the preparation of mapping primers. Outputs will be the identification of three genes and the lesions in the mutant gene copies. 2. Localize the gene products of the ORY1, COR1, and SUPCS1 genes. Numerous possibilities exist for products of the genes, including microtubule-associated proteins and proteins involved in cell cycle control. Recently, oryzalin was shown to cause changes in the morphology of both ER and Golgi in Arabidopsis roots and other plant cells (Langhans et al., 2009), suggesting that the gene products function in these compartments of the endomembrane system. It is possible that the genes that are the focus of this proposal also function in protein folding. The synthetic lethal interaction between the ory1 and cor1 mutations may reflect a physical interaction of the gene products or may indicate that the products perform a similar function. Similarly, the suppression of ory1 phenotypes by supcs1 may indicate that the products of these genes interact or function in the same pathway. Identification of the three genes of interest will likely provide an indication of gene function based on evolutionary conservation and annotation of the genes and will inform the types of experiments we carry out to determine the cellular localization of the proteins. It is also possible that these genes will encode "novel" proteins with no known function and that cellular localization of the proteins will be very important for elucidating gene function. Outputs will be information on the cellular localization of the proteins of interest. 3. Expand the network of genes. We will use methods that were previously successful for identifying new genes involved in a genetic process by isolating mutants that suppress the phenotype of a starting gene. For example, the supcs1 mutation was identified as an extragenic suppressor of temperature-sensitivity in ory1 mutants. We isolated more than 25 extragenic suppressors of ory1 (James et al., 1989), of which 15 had phenotypes in the absence of the ory1 mutation, including colchicine and/or taxol resistance, oryzalin super-sensitivity and cold-sensitivity. We will focus on suppressors of the ory1 mutation and the supcs1 mutation. Outputs will be the identification of other genes that have genetic interactions with the genes that are the focus of this proposal.
Project Methods
1. Positional cloning of the ORY1, COR1, and SUPCS1 genes ORY1 gene. Mutations in the ORY1 gene confer resistance to oryzalin, colchicine, and taxol and have a temperature-sensitive lethal phenotype at 34 degrees C. (James et al., 1989). The ORY1 locus is on chromosome 10, located 12.3 cM from pf24. A double mutant ory1 pf24 strain will be crossed to the polymorphic S1D2 strain to obtain ory1 and PF24 recombinant progeny. DNA from the recombinant strains will be tested using SNP markers to identify the recombination point most closely linked to ORY1. BAC clones containing wild-type DNA covering the genomic region of the ORY1 gene will be transformed into the ory1 strain to test for rescue of the mutant phenotype. Genes located on the BAC clone capable of rescuing the phenotype will be candidates for ORY1. Sequences of candidate genes from the ory1 strains will be compared with wild-type sequences to identify the lesions in the ory1 alleles. A cloned wild-type gene will be tested for transformation rescue of the phenotype to confirm the identity of the ORY1 gene. COR1 gene. The cor1 mutant strain shows ~3-fold resistance to colchicine compared to wild-type cells and a temperature-sensitive lethal phenotype at 34 degrees C. (James et al., 1989). The cor1 and ory1 mutations show a synthetic lethal interaction. The COR1 locus maps to chromosome 12, between the ODA6 and NIC15 loci. We will identify the COR1 gene using the approach described above. SUPCS1 gene. The supcs1 mutation suppresses the temperature-sensitive phenotype of the ory1 mutation and, on its own, has a phenotype of cold-sensitive growth at 15o. The gene maps to chromosome 3, less than 0.2 cM from the TUA1 gene (James, 1989). Using TUA1 as a starting point, we will prepare DNA from a tiling set of BAC clones extending on either side of TUA1 and identify the TUA1 gene using methods described above. 2. Localize the gene products of the ORY1, COR1, and SUPCS1 genes. We will prepare tagged gene constructs for localization studies using a 3X HA tag (Silflow et al., 2001; Piasecki et al., 2008). The constructs will be tested for their ability to rescue the mutant phenotype in transformation experiments. Phenotypically rescued cells will be examined using immunofluorescence microscopy to determine the localization of the tagged protein. For co-localization studies, we will use polyclonal antibodies against tubulin, ER proteins and Golgi proteins (Agrisera Company), and Mitotracker orange for localization of mitochondria (Yoshihara et al., 2008). 3. Expand the network of genes involved in regulating microtubules We will isolate and characterize new extragenic suppressor mutations of ory1 by screening for growth at 33 degrees C, and of supcs1 by screening for growth at 16 degrees C. Revertant colonies that grow at the non-permissive temperature will be backcrossed to wild-type cells, and extragenic suppressor mutations that demonstrate a drug-resistance or temperature-conditional phenotype in the absence of the starting mutation will be analyzed further. We will use next generation Illumina sequencing to identify the genetic lesion in the extragenic suppressor mutants.