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Comparison of wild-type and cell death mutant of barley plants containing Mla6 powdery mildew resistance gene

ABSTRACT: Time-course expression profiles of Bgh challenged barley cultivar C.I. 16151 (harboring the Mla6 powdery mildew resistance allele) and its fast-neutron-derived "Bgh-induced tip cell death1" mutant, bcd1, were compared using the 22K Barley1 GeneChip. Planting, stage of seedlings, harvesting, and experimental design were part of a larger experiment described by Caldo et al. (2004). PLEXdb BB4. Experiment Design: C.I. 16151 (wildtype) and bcd1 (mutant) were planted in separate 20 x 30-cm flats using sterilized potting soil. Each experimental flat consisted of six rows of 15 seedlings, with rows randomly assigned to one of six harvest time points (0, 8, 16, 20, 24, and 32 hai). Seedlings grown to the 1st leaf stage with 2nd leaf unfolded were inoculated with a high density of fresh conidiospores (84 +/- 19 spores/mm2). Groups of flats were placed at 18C (8-hour darkness, 16-hour light) in separate controlled growth chambers corresponding to the Bgh isolates. Rows of plants were harvested at each assigned time points and snap frozen in liquid nitrogen. The entire experiment was repeated three times in a standard split-split-plot design with 72 experimental units (2 genotypes x 2 pathogen isolates x 6 time points x 3 replications). Treatment Description: The samples constituted pairwise combinations of the the cultivar C.I. 16151(containing the Mla6 resistance allele), and its fast-neutron-derived "Bgh-induced tip cell death1" mutant, bcd1 with the two Bgh (Blumeria graminis f. sp. hordei) isolates, 5874 (AvrMla6, AvrMla1) and K1 (AvrMla13, AvrMla1). For each replication, individual genotypes were planted in separate 20 x 30 cm flats using sterilized potting soil. Each experimental flat consisted of six rows of 15 seedlings, with rows randomly assigned to one of six harvest times (0, 8, 16, 20, 24, and 32 hai). Seedlings were grown to the 2nd-leaf stage with 1st leaf unfolded, and inoculation was performed at 4 PM Central Standard Time by tipping the flats at 45oC and dusting the plants with a high density of fresh conidiospores [84 +/- 19 spores/mm2]. This procedure was repeated from the opposite angle to ensure that a high proportion of the cells are in contact with the fungus. This conidial density per unit leaf area routinely results in greater than 50% of epidermal cells that are successfully infected. Groups of flats were placed at 18oC (8 hours darkness, 16 hours light, 8 hours darkness) in separate controlled growth chambers corresponding to the Bgh isolate. Rows of plants were harvested at their assigned harvest times and flash-frozen in liquid nitrogen. ****[PLEXdb(http://www.plexdb.org) has submitted this series at GEO on behalf of the original contributor, Roger P Wise. The equivalent experiment is BB46 at PLEXdb.]

ORGANISM(S): Hordeum vulgare

PROVIDER: GSE14930 | GEO | 2009/02/25

SECONDARY ACCESSION(S): PRJNA111921

REPOSITORIES: GEO

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Project description:Time-course expression profiles of Bgh challenged barley cultivar C.I. 16151 (harboring the Mla6 powdery mildew resistance allele) and its fast-neutron-derived "Bgh-induced tip cell death1" mutant, bcd1, were compared using the 22K Barley1 GeneChip. Planting, stage of seedlings, harvesting, and experimental design were part of a larger experiment described by Caldo et al. (2004). PLEXdb BB4. Experiment Design: C.I. 16151 (wildtype) and bcd1 (mutant) were planted in separate 20 x 30-cm flats using sterilized potting soil. Each experimental flat consisted of six rows of 15 seedlings, with rows randomly assigned to one of six harvest time points (0, 8, 16, 20, 24, and 32 hai). Seedlings grown to the 1st leaf stage with 2nd leaf unfolded were inoculated with a high density of fresh conidiospores (84 +/- 19 spores/mm2). Groups of flats were placed at 18C (8-hour darkness, 16-hour light) in separate controlled growth chambers corresponding to the Bgh isolates. Rows of plants were harvested at each assigned time points and snap frozen in liquid nitrogen. The entire experiment was repeated three times in a standard split-split-plot design with 72 experimental units (2 genotypes x 2 pathogen isolates x 6 time points x 3 replications). Treatment Description: The samples constituted pairwise combinations of the the cultivar C.I. 16151(containing the Mla6 resistance allele), and its fast-neutron-derived "Bgh-induced tip cell death1" mutant, bcd1 with the two Bgh (Blumeria graminis f. sp. hordei) isolates, 5874 (AvrMla6, AvrMla1) and K1 (AvrMla13, AvrMla1). For each replication, individual genotypes were planted in separate 20 x 30 cm flats using sterilized potting soil. Each experimental flat consisted of six rows of 15 seedlings, with rows randomly assigned to one of six harvest times (0, 8, 16, 20, 24, and 32 hai). Seedlings were grown to the 2nd-leaf stage with 1st leaf unfolded, and inoculation was performed at 4 PM Central Standard Time by tipping the flats at 45oC and dusting the plants with a high density of fresh conidiospores [84 +/- 19 spores/mm2]. This procedure was repeated from the opposite angle to ensure that a high proportion of the cells are in contact with the fungus. This conidial density per unit leaf area routinely results in greater than 50% of epidermal cells that are successfully infected. Groups of flats were placed at 18oC (8 hours darkness, 16 hours light, 8 hours darkness) in separate controlled growth chambers corresponding to the Bgh isolate. Rows of plants were harvested at their assigned harvest times and flash-frozen in liquid nitrogen. ****[PLEXdb(http://www.plexdb.org) has submitted this series at GEO on behalf of the original contributor, Roger P Wise. The equivalent experiment is BB46 at PLEXdb.] genotype: Mla6 - pathogen isolates: 5874 - time: 0(3-replications); genotype: Mla6 - pathogen isolates: 5874 - time: 8(3-replications); genotype: Mla6 - pathogen isolates: 5874 - time: 16(3-replications); genotype: Mla6 - pathogen isolates: 5874 - time: 20(3-replications); genotype: Mla6 - pathogen isolates: 5874 - time: 24(3-replications); genotype: Mla6 - pathogen isolates: 5874 - time: 32(3-replications); genotype: Mla6 - pathogen isolates: K1 - time: 0(3-replications); genotype: Mla6 - pathogen isolates: K1 - time: 8(3-replications); genotype: Mla6 - pathogen isolates: K1 - time: 16(3-replications); genotype: Mla6 - pathogen isolates: K1 - time: 20(3-replications); genotype: Mla6 - pathogen isolates: K1 - time: 24(3-replications); genotype: Mla6 - pathogen isolates: K1 - time: 32(3-replications); genotype: 11542 - pathogen isolates: 5874 - time: 0(3-replications); genotype: 11542 - pathogen isolates: 5874 - time: 8(3-replications); genotype: 11542 - pathogen isolates: 5874 - time: 16(3-replications); genotype: 11542 - pathogen isolates: 5874 - time: 20(3-replications); genotype: 11542 - pathogen isolates: 5874 - time: 24(3-replications); genotype: 11542 - pathogen isolates: 5874 - time: 32(3-replications); genotype: 11542 - pathogen isolates: K1 - time: 0(3-replications); genotype: 11542 - pathogen isolates: K1 - time: 8(3-replications); genotype: 11542 - pathogen isolates: K1 - time: 16(3-replications); genotype: 11542 - pathogen isolates: K1 - time: 20(3-replications); genotype: 11542 - pathogen isolates: K1 - time: 24(3-replications); genotype: 11542 - pathogen isolates: K1 - time: 32(3-replications)

Project description:Stripe rust, caused by Puccinia striiformis f. sp. tritici, is a destructive disease of wheat worldwide. Genetic resistance is the preferred method for controlling stripe rust, of which two major types are race-specific and race non-specific resistance. Race-specific resistance includes the qualitatively inherited all-stage resistance, controlled by single major resistance (R) genes. Conversely, adult-plant resistance is race non-specific, inherited quantitatively, and is durable. Previously, we characterized the gene expression signatures involved in Yr5-controlled all-stage resistance and Yr39-controlled adult-plant resistance using the Affymetrix Wheat GeneChip. For this study, we designed and constructed custom oligonucleotide microarrays containing probes for the 116 and 207 transcripts that we had found important for the Yr5 and Yr39 resistance responses, respectively. We used this custom microarray to profile the resistance responses of eight wheat genotypes with all-stage resistance (Yr1, Yr5, Yr7, Yr8, Yr9, Yr10, Yr15, and Yr17). The aim of this analysis was to identify common and unique gene expression signatures involved in race-specific resistance accross genotypes, which were used to infer information regarding the general pathways involved in all-stage resistance. Keywords: Stress response Eight wheat genotypes with defined single gene all-stage resistance to particular stripe rust (Pst) isolates were selected for this study, as well as the Pst susceptible genotype âAvocet Susceptibleâ (AVS). Near Isogenic Lines (NILs) for each of eight Yr resistance genes (Yr1, Yr5, Yr7, Yr8, Yr9, Yr10, Yr15 and Yr17) were developed at the Plant Breeding Institute, Sydney, Australia, by backcrossing the Yr gene donors with the recurrent susceptible spring wheat genotype (Triticum aestivum L.) AVS six times (AVS*6/Yr*) and selecting for the appropriate resistance at each generation. Both virulent and avirulent isolates of Pst were selected for each NIL, except for Yr15, for which no virulent isolate is currently known. Care was taken to select the fewest isolates needed to satisfy the spectrum of virulence/avirulence required, resulting in the use of six isolates identified as PST-17, PST-21, PST-43, PST-45, PST-78 and PST-AUS. Each isolate was selected and maintained on susceptible genotypes. For each of three biological replications, individual genotypes were planted in separate 25 X 42.5-cm flats using a potting mix (6 peat moss: 4 vermiculite with lime: 3 sand: 3 commercial potting mix: 2 perlite: 1.7 g/L lime: 3.3 g/L Osmocote: 2.2 g/L ammonium nitrate). Each flat consisted of three rows of six seedlings, with rows randomly assigned one of two harvest times (24 and 48 h post-inoculation). Seedlings from the 3rd row were used to monitor the expected disease responses to inoculation. Seedlings were grown to the second leaf stage (Feekes 1.2, emergence with second leaf unfolded, ~10 days after planting) in a greenhouse with a diurnal temperature cycle of 10ÂºC (2:00 am) to 25ÂºC (2:00 pm) and a 16 h light/8 h dark cycle. Inoculation was performed by misting the plants with sterile water and applying a 1:20 urediniospore:talc mixture to leaves with a sterile brush. Talc was used to aid in the spread and adhesion of spores over leaf surfaces. Control flats were treated the same way except for the absence of spores in the talc. All treatments for each biological replication were performed at 9 am Pacific Standard Time. To promote spore germination, all flats were transferred to a dew chamber (100% RH) operating at 10ÂºC in the dark for 24 h, before being placed in a growth chamber with a diurnal temperature cycle of 4ÂºC (2:00 am) to 20ÂºC (2:00 pm) and a 16 h light/8 h dark cycle. Rows of plants were harvested from all flats at the assigned times for RNA extraction.

Project description:The Atss3 mutant and WT plants were arranged according to a Randomized Complete Block Design. The plants were planted in rows with seven rows in each flat; two plants of the same genotype/pot. Plants were grown under a SD photoperiod (8 h light/16 h dark) in a growth chamber as described. Eight randomly selected rows were harvested for each time point from different flats. Plant material was harvested at five time points in the diurnal cycle (1, 4, 8.5, 12, and 16 h; Time 0 is the beginning of the light period); harvesting was conducted under a green safety light. Each sample consisted of rosette leaves (leaves 5 to 8, staged following Bowmann (1994); photosynthetically active (Stessman et al., 2002)) from sixteen six-week-old plants. Leaf samples were frozen in liquid N2 immediately after harvest and stored at -80ï°C for RNA extraction. The experiment was done twice and independent randomizations for plant growth and harvest were used for the two replicates. Experiment Overall Design: The Atss3 mutant and WT plants were arranged according to a Randomized Complete Block Design. The plants were planted in rows with seven rows in each flat; two plants of the same genotype/pot. Plants were grown under a SD photoperiod (8 h light/16 h dark) in a growth chamber as described. Eight randomly selected rows were harvested for each time point from different flats. Plant material was harvested at five time points in the diurnal cycle (1, 4, 8.5, 12, and 16 h; Time 0 is the beginning of the light period); harvesting was conducted under a green safety light. Each sample consisted of rosette leaves (leaves 5 to 8, staged following Bowmann (1994); photosynthetically active (Stessman et al., 2002)) from sixteen six-week-old plants. Leaf samples were frozen in liquid N2 immediately after harvest and stored at -80ï°C for RNA extraction. The experiment was done twice and independent randomizations for plant growth and harvest were used for the two replicates.

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Comparison of wild-type and cell death mutant of barley plants containing Mla6 powdery mildew resistance gene

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