ena-STUDY-SYNGENTA / RWTH Aachen University-10-08-2021-15:01:19:316-1026
Genome and transcriptome sequencing of Asian Soybean Rust, Phakopsora pachyrhizi
High-molecular weight DNA was extracted from germinated urediniospores of Phakopsora pachyrhizi isolate K8108 using a carboxyl-modified magnetic bead protocol. The genome was sequenced on a PacBio Sequel (Pacific Biosciences, Menlo Park, CA) using a 20-kb PacBio SMRTbell library prepared by Genewiz (South Plainfield, NJ) with 15-kb Blue Pippin size selection. 69 Gbp of raw sequencing data was generated. The susceptible soybean cultivar Williams 82 was used for in planta RNA profiling. First trifoliate leaves of 19-to-21-day old soybean plants were inoculated with a 2 mg.ml-1 spore suspension. Leaf samples were harvested at 10, 24, 72 and 192 hpi. Parallel sampling was performed from mock-inoculated plants at the same timepoints. RNA from on planta fungal penetration structures were obtained following 8h incubation at 22°C under saturated humidity in the dark using the following protocol.  Liquid latex (semi-transparent low ammonium, Latex-24, Germaringen, Germany) was sprayed (hand spray gun with gas unit, Preval, Bridgeview, USA) until complete leaf coverage. After drying off, latex was removed. These samples contain the appressoria and spores from the leaf surface but no plant tissue. Latex samples from a single middle leaflet from three separate plants were bulked for each sample used for RNA extraction.  RNA from in vitro germinating spores were obtained using polyethylene foil (dm freezer bag, Karlsruhe, Germany) placed in glass plates and inoculated with a spore suspension (2 mg ml-1). Each biological replicate corresponded to 500 cm² foil and ~4 mg urediniospores. The plates were incubated at 22°C in the dark at saturated humidity for 0.5, 2, 4 or 8 h. After incubation, the spores were collected using a cell scraper. RNA from spores germinated in the presence of in vitro antifungal SDHI stress were collected from 5 ml of spore suspension (1 mg ml-1 in 0.01 % (vol/vol) Tween-20 in distilled water). For the treated samples, 2.5 ul of a 1 mM benzovindiflupyr stock solution in 1 % (v/v) DMSO was added to a final concentration of 0.5 µM benzovindiflupyr. A 1% (v/v) DMSO solution was used for the control samples. Spores were gently shaken for 4 h at room temperature in the dark and then collected by centrifugation (3220 g for 15 min). All RNA preparations were subjected to a final DNase treatment and purification with the RNeasy Mini purification kit (Qiagen, Hilden, Germany). Quantity and quality of the RNAs were assessed using a TapeStation instrument (Agilent, Santa Clara, CA). 1 µg per sample was used to prepare the library using the NEBNext® Ultra™ II RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA). Libraries were then normalized to 10mM, pooled and sequenced at 150-bp paired-end on the HiSeq X instrument at Genewiz (South Plainfield, NJ), with ten samples per lane.
High-molecular weight DNA was extracted from germinated urediniospores of Phakopsora pachyrhizi isolate K8108 using a carboxyl-modified magnetic bead protocol. The genome was sequenced on a PacBio Sequel (Pacific Biosciences, Menlo Park, CA) using a 20-kb PacBio SMRTbell library prepared by Genewiz (South Plainfield, NJ) with 15-kb Blue Pippin size selection. 69 Gbp of raw sequencing data was generated. The susceptible soybean cultivar Williams 82 was used for in planta RNA profiling. First trifoliate leaves of 19-to-21-day old soybean plants were inoculated with a 2 mg.ml-1 spore suspension. Leaf samples were harvested at 10, 24, 72 and 192 hpi. Parallel sampling was performed from mock-inoculated plants at the same timepoints. RNA from on planta fungal penetration structures were obtained following 8h incubation at 22°C under saturated humidity in the dark using the following protocol.  Liquid latex (semi-transparent low ammonium, Latex-24, Germaringen, Germany) was sprayed (hand spray gun with gas unit, Preval, Bridgeview, USA) until complete leaf coverage. After drying off, latex was removed. These samples contain the appressoria and spores from the leaf surface but no plant tissue. Latex samples from a single middle leaflet from three separate plants were bulked for each sample used for RNA extraction.  RNA from in vitro germinating spores were obtained using polyethylene foil (dm freezer bag, Karlsruhe, Germany) placed in glass plates and inoculated with a spore suspension (2 mg ml-1). Each biological replicate corresponded to 500 cm² foil and ~4 mg urediniospores. The plates were incubated at 22°C in the dark at saturated humidity for 0.5, 2, 4 or 8 h. After incubation, the spores were collected using a cell scraper. RNA from spores germinated in the presence of in vitro antifungal SDHI stress were collected from 5 ml of spore suspension (1 mg ml-1 in 0.01 % (vol/vol) Tween-20 in distilled water). For the treated samples, 2.5 ul of a 1 mM benzovindiflupyr stock solution in 1 % (v/v) DMSO was added to a final concentration of 0.5 µM benzovindiflupyr. A 1% (v/v) DMSO solution was used for the control samples. Spores were gently shaken for 4 h at room temperature in the dark and then collected by centrifugation (3220 g for 15 min). All RNA preparations were subjected to a final DNase treatment and purification with the RNeasy Mini purification kit (Qiagen, Hilden, Germany). Quantity and quality of the RNAs were assessed using a TapeStation instrument (Agilent, Santa Clara, CA). 1 µg per sample was used to prepare the library using the NEBNext® Ultra™ II RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA). Libraries were then normalized to 10mM, pooled and sequenced at 150-bp paired-end on the HiSeq X instrument at Genewiz (South Plainfield, NJ), with ten samples per lane.