Identification of the wheat TaClpS1
In this study, wheat TaClpS1 was identified using the protein sequence of Arabidopsis AtClpS1 (GenBank accession no. NP_564937.1) blasting the hexaploid wheat genome databases (http://plants.ensembl.org/index.html). The results showed that six homologous sequences of AtClpS1 in wheat were located on chromosomes 2A, 2B, 2D, 3A, 3B and 3D, respectively. Phylogenetic analysis of the ClpS1 proteins from various plant species showed that the TaClpS1-2A, TaClpS1-2B, and TaClpS1-2D proteins are closely related to ClpS1 proteins from other plants, including Arabidopsis, Zea mays and Oryza sativa. The TaClpS1H-3A, TaClpS1H-3B, and TaClpS1H-3D proteins are not included in this group (Fig. 1A). Therefore, in this study we focused on the function of TaClpS1-2A, TaClpS1-2B, and TaClpS1-2D.
The predicted proteins TaClpS1-2A, TaClpS1-2B, and TaClpS1-2D all encoded 161 amino acids, with a sequence identity of 98.77%. Sequences of the predicted proteins TaClpS1-2A, TaClpS1-2B, and TaClpS1-2D were determined to contain a conserved ClpS domain by using Pfam online (http://pfam.xfam.org/) (Fig. 1B). Based on the above analyses, it was concluded that the identified genes TaClpS1-2A, TaClpS1-2B, and TaClpS1-2D encode the ClpS1 protein in wheat.
TaClpS1 is localized in the chloroplast of wheat
To determine the subcellular location of TaClpS1, using localizer online (http://localizer.csiro.au/), TaClpS1-2A, TaClpS1-2B, and TaClpS1-2D were predicted to contain a chloroplast transit peptide (1-32 aa) (Fig. 1B). Considering that TaClpS1-2A, TaClpS1-2B, and TaClpS1-2D are highly conserved in amino acid sequence, TaClpS1-2A was selected as a representative of all TaClpS1 and generated the fusion constructs pCAMBIA1302: TaClpS1–GFP. TaClpS1-2A lacking the chloroplast transit peptide (1-32 aa) (TaClpS1Δ) was fused into vector pCAMBIA1302 to generate pCAMBIA1302: TaClpS1Δ–GFP, which was used as a negative control. These constructs were transformed into N. benthamiana leaves via A. tumefaciens infiltration. Confocal microscopy showed that TaClpS1–GFP was localized in the nucleus, cytomembrane and chloroplast of N. benthamiana, while control pCAMBIA1302: GFP and pCAMBIA1302: TaClpS1Δ–GFP were localized in the nucleus, cytomembrane and cytoplasm (Fig. 2A).
To further confirm the localization of TaClpS1 in wheat cells, the fusion constructs pCaMV35S: TaClpS1-GFP and pCaMV35S: TaClpS1Δ-GFP were generated and transformed into wheat protoplasts by polyethyleneglycol (PEG)-calcium. GFP fluorescence signals of pCaMV35S: TaClpS1Δ-GFP and pCaMV35S: GFP appeared in the nucleus, cytomembrane and cytoplasm of wheat protoplasts. In contrast to the results observed in N. benthamiana leaves, GFP fluorescence signals of TaClpS1-GFP aggregated mainly in chloroplasts of wheat protoplasts (Fig. 2B).
Relative transcript levels of TaClpS1 at different stages during Pst infection
To explore the role of TaClpS1 during Pst infection, the qRT-PCR assay was performed to examine the relative transcript levels of TaClpS1 at different stages during infection by Pst isolate CYR23. qRT-PCR data showed that the transcript levels of TaClpS1 increased as early as 12 h post-inoculation (hpi), continued to increase to 24 hpi, and subsequently diminished at 48 hpi, before the transcript levels rose again at 96 and 120 hpi (Fig. 3). The qRT-PCR results clearly indicate that the transcript levels of TaClpS1 in wheat were induced during Pst infection, suggesting that TaClpS1 participates in the interaction between wheat and Pst.
TaClpS1 is a negative regulator of wheat resistance to Pst
To examine whether TaClpS1 is involved in regulating the wheat defense resistance against Pst, the Barley Stripe Mosaic Virus (BSMV)-induced gene silencing (VIGS) strategy was used. Two specific fragments (TaClpS1-1/2as) were designed to specifically silence all three copies of the endogenous TaClpS1 gene (TaClpS1-2A/2B/2D) in Su11 wheat (Additional file 1: Fig. S1). All the wheat leaves inoculated with BSMV: γ (negative control) or BSMV: TaClpS1-1/2as displayed mild chlorotic mosaic symptoms at 12 dpi (Fig. 4A). Subsequently, the fourth leaves of silenced wheat were inoculated with incompatible Pst isolate CYR23. The leaves inoculated with CYR23 displayed hypersensitive response (HR) symptoms in negative controls and BSMV: TaClpS1-1/2as silenced lines (Fig. 4A). qRT-PCR confirmed that the transcript levels of TaClpS1 were significantly reduced in BSMV: TaClpS1-1/2as silenced lines compared with BSMV: γ treated wheat at 0, 24 and 120 hpi (Fig. 4B). Pathogenesis-related (PR) genes, including PR1 and PR2, are generally considered as marker genes in HR and are necessary for resistance of plants to pathogens [21, 22]. Then, the transcript levels of TaPR1 and TaPR2 were analyzed in TaClpS1-1/2as silenced lines and BSMV: γ treated wheat inoculated with CYR23 at 0, 24 and 120 hpi. Our qRT-PCR results showed that the transcript levels of TaPR1 (Fig. 4C) and TaPR2 (Fig. 4D) were notably increased in TaClpS1-1/2as silenced lines compared with that in BSMV: γ treated wheat. In addition, the areas of necroses and H2O2 accumulation induced by inoculation with CYR23 in wheat leaves were measured at 24 hpi. As shown in Fig. 5, H2O2 accumulation per infection site (Fig. 5A, B) and the necrotic area (Fig. 5C, D) in TaClpS1-1/2as silenced wheat were obviously greater than that in BSMV: γ treated wheat. Taken together, these results revealed that TaClpS1 stimulates the Pst infection in wheat, which finally increased plant susceptibility during wheat-Pst incompatible interaction.
Silencing TaClpS1 significantly inhibits the growth of Pst
In addition to analyze necroses and H2O2 accumulation, histological analysis of mycelial structures of Pst was performed in wheat leaves infected with CYR23 at 24 and 120 hpi (Fig. 6A). The numbers of haustoria (Fig. 6B), hyphal lengths (Fig. 6C) and hyphal infection area (Fig. 6D), which are indicators to assess fungal expansion ability, were strictly reduced in TaClpS1-1/2as silenced wheat compared with that in BSMV: γ treated wheat. These results indicated that silencing TaClpS1 diminished the growth of Pst.
TaClpS1 negatively regulates disease resistance of N. benthamiana to Phytophthora parasitica
To further verify the conclusion that TaClpS1 negatively regulates plant resistance against pathogens, firstly the interaction of the model plant N. benthamiana and oomycete Phytophthora parasitica was examined. In this experiment, A. tumefaciens carrying plasmid pCAMBIA1302: TaClpS1–GFP or pCAMBIA1302: GFP (negative control) were infiltrated, and then P. parasitica mycelial plugs were placed onto the infiltrated N. benthamiana leaves. As expected, compared with the pCAMBIA1302: GFP negative control, lesion diameters of leaves expressing pCAMBIA1302: TaClpS1–GFP were significantly larger, demonstrating that ectopic expression of TaClpS1 in N. benthamiana can enhance P. parasitica infection (Fig. 7A, B). In addition, VIGS strategy was utilized to silence TaClpS1 and then inoculated with Pst virulent isolate CYR31. As shown in Fig. 7C, the uredia on TaClpS1 silenced leaves inoculated with CYR31 at 12 hpi were less than that on negative controls. Moreover, qRT-PCR confirmed that the transcript levels of TaClpS1 were significantly reduced in BSMV: TaClpS1-1/2as silenced lines compared with BSMV: γ treated wheat at 24 and 120 hpi (Fig. 7D). Taken together, these results indicate that TaClpS1 negatively regulates disease resistance of plants.
TaClpS1 interacts with TaHEMA1 by yeast two-hybrid assay
In Arabidopsis, GluTR, encoded by gene HEMA1 (AT1G58290), was identified as a candidate substrate of AtClpS1 . To confirm whether glutamyl-tRNA reductase is a substrate of TaClpS1 in wheat, yeast two-hybrid (Y2H) technique was used. Firstly, a BLAST search using the protein sequence of Arabidopsis HEMA1 as a query showed that the hexaploid wheat genome contains three homologous sequences of AtHEMA1, which were located on chromosomes 1A, 1B and 1D, respectively. Subsequently, the three homologous genes were named TaHEMA1 based on phylogenetic tree constructed with HEMA1 proteins from various plant species (Additional file 2: Fig. S2A). Considering that the three copies of TaHEMA1 are highly conserved in amino acid sequence with 98.37% identity (Additional file 2: Fig. S2B), TaHEMA1 in chromosomes 1B was selected as a representative to perform Y2H assay. In Y2H assay, only yeast cell carrying TaHEMA1 and TaClpS1 could grow normally on SD-Leu-Trp-His-Ade medium containing X-α-gal and appeared blue (Additional file 2: Fig. S2C), indicating that TaClpS1 interacts with TaHEMA1. Overall, these results suggest GluTR encoded by TaHEMA1 could be a candidate substrate of TaClpS1 in wheat.