Strains, plant materials and growth
In this study, Pst race CYR23 and CYR31 was used to investigate the transcript levels of TaClpS1 and the VIGS assay of TaClpS1 according to the procedure described previously . Fresh Pst urediospores were collected from wheat infected with Pst. P. parasitica strain ZQ-1 used in this study was routinely maintained on 10% V8 juice medium at 25 °C in the dark.
Wheat (Triticum aestivum L.) variety Suwon11 (AUS-22519) originating from Seuseun Agricultural Experiment Station (Sariwon, Korea) was registered in the Australian Winter Cereal Collection, Tamworth, Australia. Suwon11, containing YrSu resistance gene , is resistant to Pst race CYR23. Suwon11 used in this study was confirmed by Qian Yang. Suwon11 seedlings were grown and maintained in an artificial climate chamber at 16 °C. Tobacco (Nicotiana benthamiana) plants were grown in growth rooms at 21-25 °C with a 16-h/8-h light/dark cycle. CYR23, Suwon11 seeds and N. benthamiana seeds were obtained from the Prof. Zhensheng Kang’s Lab (Northwest A&F University, China). P. parasitica strain ZQ-1 was obtained from Prof. Yongli Qiao (Shanghai Normal University, China).
The full length of TaClpS1 was cloned into T-simple19 vector to generate TaClpS1-T construct from wheat cultivar Suwon11 cDNA with TaClpS1-specific primers TaClpS1-F/R (Additional file 2: Table S1). To create the constructs for examining the subcellular localization of TaClpS1, full-length TaClpS1 and TaClpS1Δ were amplified from the above TaClpS1-T construct and inserted into pCAMBIA1302 or pTF486 vector  to generate pCAMBIA1302: TaClpS1–GFP, pCAMBIA1302: TaClpS1Δ–GFP and pCaMV35S: TaClpS1-GFP, pCaMV35S: TaClpS1Δ-GFP respectively. Primer sequences are shown in Additional file 2: Table S1. For VIGS assay, two approximately 150-bp specific silencing fragments were designed based on the combination of Primer5 and NCBI. The two designed fragments were cloned and inserted into BSMV: γ vector to prepare recombinant plasmids BSMV: TaClpS1-1as and BSMV: TaClpS1-2as using specific primers shown in Additional file 2: Table S1. Above all constructs were obtained from the Prof. Zhensheng Kang’s Lab (Northwest A&F University, China).
For phylogenetic analysis of TaClpS1, Clps1 proteins from other plants were obtained using the protein sequence of Arabidopsis AtClpS1 (GenBank accession no. NP_564937.1) to blast NCBI databases. The phylogenetic tree of ClpS1 proteins from plants was constructed based on the maximum-likelihood method using MEGA5 software. The conserved ClpS domain was analyzed using Pfam online (http://pfam.xfam.org/).
RNA extraction and analyses of transcript levels
The second leaves of the two-leaf stage wheat seedlings were inoculated with Pst race CYR23. After inoculation, three independent wheat leaves were sampled at 0, 12, 24, 48, 72, 96, 120 hpi for extracting RNA. Total RNA was extracted with the Quick RNA isolation Kit (Huayueyang Biotechnology, China, Beijing). About 3 μg of the total extracted RNA was used for reverse transcription to cDNA with RevertAid First Strand cDNA Synthesis Kit. For RNA extraction and reverse transcription in VIGS assay of TaClpS1, the methods were as described above. LightCycler SYBR Green I Master Mix was used for the qRT-PCR assay, and the transcript levels of genes were normalized to the internal control gene TaEF-1α. The primers used in qRT-PCR assay are listed in Additional file 2: Table S1. The statistical significance was evaluated by unpaired two-tailed Student’s t-test.
Subcellular localization analysis
To determine the subcellular localization of TaClpS1 in N. benthamiana leaves, A. tumefaciens carrying pCAMBIA1302: TaClpS1–GFP, pCAMBIA1302: TaClpS1Δ–GFP or pCAMBIA1302: GFP vector at a final OD600 of 0.5 was infiltrated into N. benthamiana leaves. Vectors pCAMBIA1302: TaClpS1Δ–GFP and pCAMBIA1302: GFP were used as negative controls. The infiltrated N. benthamiana were maintained in growth rooms at 21-25 °C with a 16-h/8-h light/dark cycle. At 48 h after agroinfiltration, confocal images were taken with an Olympus IX83 confocal microscope (Japan), using excitation and emission wavelengths of 488 and 520nm for GFP and 561 and 640 nm for autofluorescence of chloroplast, respectively.
For testing the subcellular localization of TaClpS1 in wheat cells, Triticum aestivum Suwon11 seedlings were grown in the glasshouse at 25 °C for 2–3 weeks for protoplast transformation. The fusion constructs pCaMV35S: TaClpS1-GFP, pCaMV35S: TaClpS1Δ-GFP and pCaMV35S: GFP were independently transformed into wheat protoplasts by polyethyleneglycol (PEG)-calcium as described previously [31, 32]. The mixtures containing pCaMV35S: TaClpS1-GFP or pCaMV35S: GFP and wheat protoplasts were incubated at 22 °C. Confocal images were taken with an Olympus IX83 confocal microscope (Japan) at 24 h after incubation, using excitation and emission wavelengths of 488 and 520nm for GFP and 561 and 640 nm for autofluorescence of chloroplast, respectively.
BSMV-mediated gene silencing
Plasmids BSMV: TaClpS1-1as, BSMV: TaClpS1-2as and BSMV: γ were linearized followed by transcribing and capping in vitro using the RiboMAX Large-Scale RNA Production System-T7 and the Ribom7G Cap Analog (both by Promega) according to the manufacturer’s instructions. Wheat leaves were inoculated with the capped BSMV transcripts and Pst race CYR23 and CYR31 according to the procedure described previously . BSMV: γ was used as the negative control. The wheat leaves infected with CYR23 were sampled at 0, 24, and 120 hpi for estimating the transcript levels of TaClpS1 and TaPR1/2, H2O2 detection, measuring necrotic areas and histological observations in VIGS assay of TaClpS1. The symptoms on the wheat leaves were photographed at 12 d after inoculation with Pst CYR23 and CYR31. These experiments were repeated at least two times.
DAB staining for H2O2 detection, measuring necroses
The wheat leaves inoculated with CYR23 in VIGS assay were sampled and stained in 1 mg/ml 3,3-diaminobenzidine (DAB) solution for 6 h at 16 °C under illumination. After staining, the leaves were clarified in the destaining solution (absolute alcohol: acetic acid glacial, 1:1) for about one week. Then, the decolored wheat leaves were further clarified with chloral hydrate for two weeks. Subsequently, H2O2 accumulation in the transparent leaves was detected with an Olympus BX-51 microscope under bright-field illumination. Alongside detection of H2O2 accumulation, necrotic areas were measured under UV channel of an Olympus BX-51 microscope. The results were obtained from 40 infection sites. The samples were collected from three independent leaves. The experiments were repeated three times. The statistical significance was evaluated by unpaired two-tailed Student’s t-test.
Histological observations of Pst growth
For histological observations of Pst growth, the wheat leaves inoculated with CYR23 in VIGS experiments were decolorized at 24 and 120 hpi in destaining solution (absolute alcohol: acetic acid glacial, 1:1) for about one week. Then, clarified wheat samples were stained with wheat germ agglutinin (WGA) conjugated to Alexa-488 (Invitrogen, USA) as described previously [34, 35]. For each biological replicate, 40 infection sites of each sample from three separate leaves were recorded to assess the number of haustoria, hyphal length and infection area. The statistical significance was evaluated by unpaired two-tailed Student’s t-test.
P. parasitica inoculation
N. benthamiana leaves infiltrated with A. tumefaciens carrying pCAMBIA1302: TaClpS1–GFP, pCAMBIA1302: GFP were detached at 36 h after infiltration and challenged with P. parasitica by placing mycelial plugs (5 mm diam) onto the detached N. benthamiana leaves. The inoculated leaves were maintained in a growth room at 25 °C in darkness. At 36 hpi, the inoculated leaves were stained with trypan blue as previously described . Stained leaves were photographed and the diameters of the lesion area were measured.