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 Protocols

Plant and Pathogen Gene Expression during Infection by Phytophthora sojae of 8 soybean (Glycine max) Cultivars Varying in Quantitative Disease Resistance

Plant Materials and Pathogen Isolate

Eight soybean genotypes with varying degrees of quantitative resistance that were well-characterized were selected for this study. These included four genotypes with high levels of QR [Athow (Ath), V71-370 (V71), General (Gen) and Conrad (Con)], two with moderate levels of resistance [Williams (Wil) and PI291327 (PI2)], and two susceptible genotypes [Sloan (Slo) and OX20-8 (OX2)]. A P. sojae isolate, PT2004C2.S1 , which is virulent to the soybean genotypes carrying Rps1a, Rps1b, Rps1k, Rps2, Rps3a, Rps3c, Rps4, Rps5, Rps6, or Rps7, was used in this study.

Experimental Design

The experiment was repeated four times. A balanced, split-plot design was used for the experiment, with the eight soybean genotypes as the whole plot factor, and inoculation treatments (pathogen-inoculated vs. mock-inoculated) and sampling time (3 and 5 days post inoculation (dpi)) as the split-plot factors. Each experimental replicate included 32 conditions, which were the complete combinations of the 8 genotypes, 2 time points, and 2 treatments. Within each experimental replicate, there were two sets of inoculation replications, each of which had a set of 30 plants.

Inoculation Assay

Inoculation assays were performed using the slant board technique (Olah and Schmitthenner, 1985).[ Briefly, 7-day-old soybean seedlings were thoroughly rinsed under tap water and placed in an inoculation tray (10 plants per tray, 3 trays per inoculation replication). The plants were wounded at 2 cm below the beginning of the root zone by scraping the epidermis with a scalpel and then inoculated with a mycelial slurry from a 7-day-old culture or agar alone. Samples of root tissues were collected at 3 and 5 days post inoculation (dpi) from 7.5 mm below and above the lesion margin from each seedling with characteristic lesions. For the mock-inoculated plants, tissue sections were taken 7.5 mm below and above the position corresponding to the average lesion length measured from the inoculated samples. The lesion length (mm) from the inoculation point up to the edge of the lesion margin was also measured 3 and 5 dpi. For each experimental replication, 2 replicates of 30 seedlings per condition were inoculated and harvested separately. All the plants for an experiment were grown in the same environmental growth chamber (Chagrin Falls, Ohio; Model M-48 with TC2 microcontroller unit) with day and night temperatures settings of 27C and 21C and relative humidity averaging 75 to 90%.

RNA Extraction

The QIAGEN RNeasy(r) Plant Mini Kit was used throughout for total RNA isolation from pathogen- or mock-inoculated soybean tissue sections. The manufacturer provided protocol for total RNA extraction from plant cells and tissues and filamentous fungi was followed with minor modifications in order to obtain a sufficient amount of high quality RNA. The quality of total RNA was checked in an Agilent 2100 Bioanalyser.

Microarray Assay

RNA samples from the two inoculation replications were pooled in equal amounts. RNA samples from mock-inoculated soybean tissue were added 2%-16% spike-in RNA from Phytophthora sojae mycelium grown in liquid sucrose-salts medium, depending on the resistance level and sampling time (see table below)

Cultivars3 dpi5 dpi
Athow,V71-370, General,Conrad(Highly resistant 2% 4%
Williams, PI291327, Sloan, OX20-8(moderately resistant or susceptible) 8% 16%

Microarray procedures were performed at the Core Laboratory Facility of Virginia Bioinformatics Institute following the standard eukaryotic gene expression assay protocols described in the Affymetrix GeneChip(r) Expression Analysis Technical Manual. In brief, the One-Cycle Target Labeling and Control Reagents (Affymetrix) and 1ug of total RNA were used to generate biotin-labeled cRNA. Twenty micrograms of labeled cRNA was fragmented in Fragmentation Buffer and then hybridized to a soybean GeneChip. Hybridization was performed at 45C for 16 h in an Affymetrix hybridization oven (model 640), GeneChips were washed and stained with streptavidin-phycoerythrin using the fluidics protocol EukGE-WS2v5-450 in the Affymetrix(r) GeneChip(r) Fluidics Station 450, and stained chips were scanned with an Affymetrix GCS3000 7G Scanner. The Affymetrix GeneChip(r) Operating Software (GCOS, v1.4.0.036) was used to provide instrument control, first-level data analysis, and data management for the entire GeneChip System.

A total of 128 soybean GeneChips were used for the entire experiment.

Temporal and spatial patterns of plant and pathogen gene expression during infection by Phytophthora sojae of 4 soybean (Glycine max) cultivars varying in quantitative disease resistance

Plant Materials and Pathogen Isolate

Four selected soybean genotypes with varying degrees of quantitative resistance that were well-characterized were selected for this study. These included two resistant genotypes (V71-370, Conrad), and two susceptible genotypes (Sloan, and VP-RIL9). A P. sojae isolate, PT2004C2.S1 , which is virulent to the soybean genotypes carrying Rps1a, Rps1b, Rps1k, Rps2, Rps3a, Rps3c, Rps4, Rps5, Rps6, or Rps7, was used in this study.

Experimental Design

The experiment was repeated four times.
Factors assayed included:

Genotype: 4 genotypes (V71-370, Conrad, Sloan, and VP-RIL9)
Treatment: pathogen-inoculated vs. mock-inoculated
Sampling time: 1, 2, 3 and 5dpi
Sampling sites: Upper section and Lower section (for 3dpi and 5dpi pathogen-inoculated samples only) vs whole section (for all the mock-inoculated samples at all time points, and the inoculated samples at 1and 2dpi).

Inoculation Assay

Inoculation assays were performed using the slant board technique (Olah and Schmitthenner, 1985). Briefly, 7-day-old soybean seedlings were thoroughly rinsed under tap water and placed in an inoculation tray (10 plants per tray, 3 trays per inoculation replication). The plants were wounded at 2 cm below the beginning of the root zone by scraping the epidermis with a scalpel and then inoculated with a mycelial slurry from a 7-day-old culture or agar alone. Samples of root tissues were collected from 7.5 mm below (for lower sections) and above (for upper sections) the lesion margin from each seedling with characteristic lesions. For the mock-inoculated plants, tissue sections were taken 7.5 mm below and above (for whole sections) the position corresponding to the average lesion length measured from the inoculated samples.
For each experimental replication, 2 replicates of 30 seedlings per condition were inoculated and harvested separately. All the plants for an experiment were grown in the same environmental growth chamber (Chagrin Falls, Ohio; Model M-48 with TC2 microcontroller unit) with day and night temperatures settings of 27C and 21C and relative humidity averaging 75 to 90%.

RNA Extraction

The QIAGEN RNeasy(r) Plant Mini Kit was used throughout for total RNA isolation from pathogen- or mock-inoculated soybean tissue sections. The manufacturer provided protocol for total RNA extraction from plant cells and tissues and filamentous fungi was followed with minor modifications in order to obtain a sufficient amount of high quality RNA. The quality of total RNA was checked in an Agilent 2100 Bioanalyser.

Microarray Assay

The RNA samples isolated from each of the two inoculation replications were pooled in equal amounts before subjecting to microarray assays. RNA samples from mock-inoculated soybean tissue contain 0.03125%-4% spike-in RNA from Phytophthora sojae mycelium grown in liquid sucrose-salts medium, depending on the resistance level and sampling time (see table below):

Genotypes1 dpi2 dpi3 dpi5 dpi
V71-370, Conrad0.03125%0.0625%0.125%0.25%
Sloan, VP-RIL90.5%1%2%4%

Microarray procedures were performed at the Core Laboratory Facility of Virginia Bioinformatics Institute following the standard eukaryotic gene expression assay protocols described in the Affymetrix GeneChip(r) Expression Analysis Technical Manual. In brief, the One-Cycle Target Labeling and Control Reagents (Affymetrix) and 1ug of total RNA were used to generate biotin-labeled cRNA. Twenty micrograms of labeled cRNA was fragmented in Fragmentation Buffer and then hybridized to a soybean GeneChip. Hybridization was performed at 45C for 16 h in an Affymetrix hybridization oven (model 640), GeneChips were washed and stained with streptavidin-phycoerythrin using the fluidics protocol EukGE-WS2v5-450 in the Affymetrix(r) GeneChip(r) Fluidics Station 450, and stained chips were scanned with an Affymetrix GCS3000 7G Scanner. The Affymetrix GeneChip(r) Operating Software (GCOS, v1.4.0.036) was used to provide instrument control, first-level data analysis, and data management for the entire GeneChip System.
A total of 160 soybean GeneChips were used for the entire experiment.