Wheat cultivar Suwon11 and Pst races CYR23 and CYR31 were used in this study. The method used for growing and inoculating of wheat seedlings were described by Kang and Li (1984). Suwon11 and CYR23 constituted an incompatible interaction that exhibited a typical HR, while Suwon11 was highly susceptible to CYR31 (Cao et al., 2003). CYR23 was maintained on wheat cv. Mingxian 169, whereas CYR31 maintained on wheat cv. Suwon 11.

To evaluate the expression levels of TaARPC3 in response to Pst infection, wheat leaves were harvested at 0, 6, 12, 18, 24, 36, 48, 72, and 120 hour post-inoculation (hpi) based on previous microscopic observations of the interactions between wheat and Pst (Wang et al., 2007). To analyze the silencing efficiency of virus-induced gene silencing (VIGS) and the relative expression of TaCAT1 and TaSOD, the fourth leaves of TaARPC3-knockdown wheat plants were sampled at 0, 24, 48, and 120 hpi following infection with Pst.

For hormone treatment, the 10-day-old wheat seedlings were sprayed with 500 μM salicylic acid (SA), 100 μM methyl jasmonate, 100 μM ethephon, 100 μM abscisic acid (ABA) and sampled at 0, 2, 6, 12, and 24 hpi. Wheat leaves inoculated with sterile distilled water were used as MOCK-inoculation controls.

Total RNA was isolated using the Biozol reagent (Invitrogen, Carlsbad, CA, United States). 3 μg of RNA aliquot of each sample was subjected to first strand cDNA synthesis using the oligo (dT)¹⁸ primer and Promega Reverse Transcription System (Promega, Madison, WI, United States). The primers (Supplementary Table S3) were designed according to the procedures as described by Livak and Schmittgen (2001) and quantization Real-Time PCR (qRT-PCR) assays were performed on an ABI-7500 system (Applied Biosystems, Foster City, CA, United States). The transcript level of TaARPC3 was quantified using the comparative 2^-ΔΔCT method (Livak and Schmittgen, 2001) with the wheat Elongation factor 1 (TaEF-1α) used as control (GenBank accession number Q03033) (Supplementary Table S3). Transcript abundance of each reaction was assessed in triplicate.

Arabidopsis thaliana actin related protein complex subunit 3 ARPC3 (Li et al., 2003; Cvrčková et al., 2004) was used to screen (in silico) the EST databases of wheat constructed in our lab (Ma et al., 2009; Wang et al., 2009, 2010). A 1091-bp unisequence (Genbank accession number AK336075.1) exhibiting high homology to the A. thaliana homolog, AtARPC3, was obtained from the cDNA library (Yu et al., 2010). Based on this sequence, we used specific primers TaARPC3-F and TaARPC3-R (Supplementary Table S3) to amplify the sequence of TaARPC3. The cloned open reading frame (ORF) was constructed into pGEM-T Easy (Promega, United States) for sequencing.

The cDNA sequence of TaARPC3 was analyzed in silico using the online BLAST¹ and the ORF Finder software² in NCBI. The amino acid sequence of TaARPC3 was analyzed with Pfam³ and ScanProsite⁴ for conserved domain identification. Multiple sequence alignments were performed using CLUSTALX2.0 (Chenna et al., 2003) and DNAMAN6.0 (Lynnon BioSoft, United States). A phylogenetic tree based on the neighbor-joining method was performed with the Mega 6 software (Tamura et al., 2007).

The S. cerevisiae diploid mutant strain Y06714 (MATa; ura3Δ0; leu2Δ0; his3Δ1; met15Δ0; YLR370c::kanMX4) and the wild type strain (WT) BY4741 (MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0) were obtained from EUROSCARF collection. The complete ORF of TaARPC3 was cloned into pDR195. The transformed cell (Δarc18+TaARPC3) was obtained by transforming the reconstructed vector (pDR195-TaARPC3) into the mutant strain Y06714. The transformed cell with empty vector pDR195 (Δarc18+empty) was used as control. Transformants were selected on SC-U medium at 30°C and validated using PCR.

To investigate the yeast cell survival under stress, the transformed cells with pDR195 (Δarc18+empty) and pDR195-TaARPC3 (Δarc18+ TaARPC3) and wild yeast cells (WT BY4741) were cultured on SC medium with 0.1mM H₂O₂, 2 M Sorbitol, 0.3 M NaCl and 0.1 M CaCl₂, respectively. Transformed cells with a starting optical density at 600 nm (OD₆₀₀) of 0.2 were cultured in yeast medium. The fluorescent dye 4′, 6-diamino-2-phenylindole (DAPI) was added to the cell suspensions at a concentration of 5 μg ml^-1 to counter-stain nuclei. The cells were stained with 0.5 μM TRITC-phalloidin to observe F-actin structures (Bereiterhahn and Kajstura, 1988). Cells were incubated at 30°C in phosphate buffer saline (PBS) for 30 min without shaking, followed by washing with sterile distilled water, then resuspended in distilled water. Cells were examined by fluorescence microscopy using an Olympus BX-53 microscope (Olympus, Corp., Tokyo) under blue light excitation by epifluorescence microscopy (excitation filter, 485 nm; dichroic mirror, 510 nm; and barrier filter, 520 nm).

The full-length cDNA of TaARPC3 was cloned into the pCaMV35S::GFP vector via XbaI/BamHI digestion and cloning. Protoplast isolation in wheat leaves was performed as previously described (Graham, 2002). The recombinant plasmid pCaMV35S:TaARPC3:GFP, empty vector pCaMV35S:GFP were transformed into wheat protoplasts by PEG4000. The transformed wheat mesophyll protoplasts were incubated in a dark chamber plates at 24°C for 12–36 h. GFP fluorescence was detected with an Olympus FV1000 confocal laser microscope equipped with a 488 nm filter (Olympus, Tokyo, Japan).

For VIGS, a 214-bp fragment of TaARPC3 with NotI and PacI (Supplementary Table S3) was derived from its coding sequence and amplified by RT-PCR to construct the BSMV:γ plasmid for gene silencing (Holzberg et al., 2002). To ensure the specificity for target gene silencing, multi-alignment and BLAST results indicated that the insert sequence of TaARPC3 was located at the non-conservative region and without off target possibility (Supplementary Figure S1 and Table S2). The BSMV RNAs were obtained in vitro from linearized plasmids γ-TaPDS, γ-TaARPC3, γ, α, β (Petty and Jackson, 1990) using the Message T7 transcription kit (Ambion, Austin, TX, United States) according to the manufacturer’s instruction. A 7.5 μl portion of each three transcripts of BSMV viruses (wild type or target gene carrying types) were mixed with 45 μl FES and directly applied by gently sliding the pinched fingers from the base to the tip five times with a gloved finger (Scofield et al., 2005; Bruun-Rasmussen et al., 2007). After inoculation with BSMV at the second leaf stage, plants were placed in the dark chamber for 24 h, and subsequently transferred to a growth chamber at 25 ± 2°C with a 16 h photoperiod and examined for symptoms. In all experiments, the recombinant virus BSMV:TaPDS was used as a positive control, negative control were inoculated with BSMV: γ. When the photo-bleaching phenotype (indicating the PDS gene silence) was observed, the fourth leaves of TaARPC3 silencing group were inoculated with urediospores of Pst race CYR23 and CYR31, respectively. The fourth leaves were collected at 24, 48, and 120 hpi for histological observation and qRT-PCR. The resistance or susceptible phenotypes were visible at 14 dpi.

Disease severity was assessed by counting the number of uredinia within standardized leaf area sections following infection with Pst. Leaves was collected randomly to avoid bias. The results were exhibited in the mean values of the number of uredinia on knock-down plants compare to the controls. Fungal biomass was analyzed by qRT-PCR as described previously (Livak and Schmittgen, 2001; Cheng et al., 2016). Genomic DNA were performed using the CTAB method (Del et al., 1989) from samples collected at 120 hpi after infected with Pst. Standard curve was generated by the plasmid carried the fragment of PsEF1 and TaEF1 as described (Liu J. et al., 2016; Liu P. et al., 2016) (Supplementary Table S3).

The host response and Pst growth in TaARPC3 know-down plants were observed by light microscopy. Stained leaf segments were fixed and cleared in ethanol/acetic acid (1:1 v/v). Auto-fluorescence of infected mesophyll cells was observed as a necrotic death area with an Olympus BX-51 microscope (Olympus Corp., Tokyo) (excitation filter, 485 nm; dichroic mirror, 510 nm; and barrier filter, 520 nm). Infection sites and lengths of infection hyphae were measured under the blue light excitation. H₂O₂ accumulation around the infection site was detected by using 3,3-diaminobenzidine (DAB; Amresco, Solon, OH, United States) staining, observed by the differential interference contrast optics. Fifty infection sites were randomly selected for each leaf segment, per treatment. Methods were taken as described previously (Wang et al., 2007), including identification of successful infection sites, hyphae staining and measurement of fungal structures.

Standard errors of deviation were calculated using Microsoft Excel. Statistical significance was assessed by a Student’s t-test (P < 0.05) using SPSS software (SPSS, Inc., Chicago, IL, United States).

A wheat 526-bp homolog of actin related protein was isolated from wheat cv. Suwon 11 by homology-based cloning and the obtained cDNA was designated as TaARPC3. Blast analysis of TaARPC3 nucleotide sequence in the Triticum aestivum cv. Chinese spring (CS) genome sequence revealed that at least three copies were present, localized on chromosome 7A, 7B, and 7D (Supplementary Figure S1A). The TaARPC3 exhibited 97.84, 92.78, and 97.97% identities with TaARPC3-7A, TaARPC3-7B, TaARPC3-7D, respectively. The predicted ORF of TaARPC3 encodes a protein of 174 amino acid with a hypothetical molecular weight of approximately 21 kDa and an isoelectric point (PI) of 6.71. A phylogenetic analysis of TaARPC3 with BdARPC3, OsARPC3, AtARPC3, ZmARPC3, NtARPC3, CaARPC3, SlARPC3, HsARPC3, BtARPC3, PtrARPC3, MScARPC3, CmARPC3, TgARPC3, and FgARPC3 resulted in that TaARPC3 clusters with ZmARPC3, BdARPC3 and OsARPC3, all of which are members of actin related proteins in monocotyledons (Figure 1A). Nucleic acid sequence analysis revealed that TaARPC3 shared 91% identity with BdARPC3 from Brachypodium distachyon. Multiple amino acid sequences alignment of TaARPC3 with BdARPC3, OsARPC3, AtARPC3, ZmARPC3, NtARPC3, CaARPC3, and SlARPC3 showed that TaARPC3 is predicted to encode proteins with the unique conserved domains of ARP2/3 complex ARPC3 (Figure 1B). These results indicate that TaARPC3 (Genbank accession number KU746328) is a typical member of the Arp2/3 complex in wheat.

A recent study demonstrated a role for the Arp2/3 complex in the actin-associated control of cell division and polarity in S. cerevisiae (Cabrera et al., 2011). TaARPC3 shared 38% similarity with arc18 (aka ARPC3) from S. cerevisiae (Supplementary Figure S1B). To evaluate the function of TaARPC3, we tested the ability of TaARPC3 to complement the arc18 mutation in yeast. To do this, the TaARPC3 coding sequence into pDR195 vector was transformed into the S. cerevisiae mutant strain Y06714 and heterologously expressed. As shown in Figure 2, S. cerevisiae cells harboring the empty vector pDR195, as well as the wild-type (untransformed) strain, BY4741 (control), showed a similar growth phenotype (i.e., rapid growth on SC medium than the mutant strain). The S. cerevisiae mutant Y06714 (aka, Δarc18) exhibited several morphological defects. As shown, mutant cells vary in size and shape when compared to the wild-type and the complemented strain. Cells were stained with TRITC-phalloidin to assess the subcellular organization of the F-actin, which facilitates observation of F-actin structures. As shown in Figure 2, staining revealed F-actin aggregation in the budding site in the complemented strain, as well as accumulation of actin dots at the septum-forming position. In the mutant strain, actin was observed as concentrated foci at the cell ends, adjacent to the developing septum. No significant difference was observed by fluorescence microscopy of DAPI-stained cells. In total, these data reveal a loss of actin cables and depolarized actin patches, suggesting that TaARPC3 may play a role in maintaining the polarized distribution of actin patches within the cell.

The Arp2/3 complex has been demonstrated to be required for cellular response to various abiotic stresses. To determine if TaARPC3 could complement the S. cerevisiae mutant phenotypes with respect to the perception of cell stress, we tested the function of TaARPC3 in S. cerevisiae under a variety of cellular stresses. As shown in Figure 3, we observed a significant increase in the growth rate of the complemented strain Y06714 compared with the S. cerevisiae arpc3 mutant carrying the empty expression vector pDR195. In SC-U media containing 0.1 mM H₂O₂ (oxidative stress), 2 M sorbitol (osmotic stress), 0.3 M NaCl (salt stress), and 0.1 M CaCl₂, similar results were observed (Figure 3 and Supplementary Figure S2). Yeast cells expressing pDR195 and pDR195-TaARPC3 grew more slowly than WT cells on stress-inducing medium, while the complemented strain containing pDR195-TaARPC3 grew at near wild-type levels (Figure 3 and Supplementary Figure S2), indicating complementation by TaARPC3. Taken together, these results indicate that the TaARPC3 participates in abiotic stress signaling.

To determine the subcellular localization of TaARPC3 in wheat, the green fluorescent protein (GFP) gene was fused with TaARPC3, and then expressed in wheat protoplasts. As showed in Figure 4, empty pCaMV35S:GFP vector, as a control, revealed a diffuse, non-specific cellular address, while cells expressing GFP-tagged TaARPC3 was localized predominantly in the nucleus and cytoplasm.

To determine the expression profiles of TaARPC3 mRNA, we next monitored the expression of TaARPC3 during the interaction between wheat and Pst using qRT-PCR. As shown in Figure 5A, during an incompatible interaction, TaARPC3 transcripts were up-regulated as early as 12 hpi, with peak expression at 18 hpi; transcript levels gradually reduced to pre-inoculation levels at 36–120 hpi. During a compatible interaction, TaARPC3 was also induced from the 12 hpi to 48 hpi; however, the transcript levels of TaARPC3 were much higher during an incompatible interaction than that during a compatible interaction at 12–24 hpi. These results support our hypothesis that TaARPC3 is likely to be associated with resistance of wheat against Pst infection.

To extend these assays, we next evaluated the expression of TaARPC3 as a function of hormone perception and signaling. To do this, wheat seedlings were treated with ethylene (ET), ABA, SA, and jasmonic acid (JA) to evaluate the possible linkages between immune signaling, susceptibility, and hormone biosynthesis. As shown in Figure 5B, when seedlings were treated with ET, the expression level of TaARPC3 was significantly induced at 12 hpi post-treatment (hpt), with an approximate 2.5-fold change in expression, after which time, transcript levels returned to levels similar to control plants (Figure 5B). After treatment with ABA, TaARPC3 transcript levels were slightly induced at up to 2 hpi, and after 2 hpi, were down-regulated (Figure 5B). Treatment with SA and MeJA was observed to down-regulate TaARPC3 expression.

The VIGS system is a rapid and effective reverse-genetic approach to characterize gene function in wheat (Yin et al., 2011). To functional the TaARPC3 in wheat-Pst interaction, we knocked down TaARPC3 using the VIGS and analyzed the impact of this on wheat response to pathogen infection. As exhibited in Figure 6A, 10 days after BSMV inoculation, mild chlorotic mosaic phenotypes were observed on the fourth leaves in BSMV-inoculated plants, with leaves inoculated BSMV:γ-PDS displaying obvious signs of photobleaching (Figure 6A); this indicates effective gene silencing. Next, the fourth leaves of wheat with BSMV-inoculated were inoculated with the avirulent race (CYR23) and virulent race (CYR31) to Suwon 11, respectively (Figure 6B). After 15 days, CYR23 elicited a pronounced HR on leaves inoculated with BSMV:γ, BSMV:TaARPC3-inocuated leaves, with uredia production observed on BSMV:TaARPC3-inocuated leaves (Figure 6B). When leaves were challenged with Pst CYR31, leaves inoculated with BSMV:TaARPC3 show more uredia produced than observed in mock plants and those inoculated with BSMV:γ (Figure 6B). To confirm silencing efficiency, the transcript level of TaARPC3 in TaARPC3 knock-down plants were evaluated by qRT-PCR. We observed that the relative expression at each time point was significantly suppressed as much as 93% (Figure 6C). Next, to quantify the disease severity in the silenced plants, fungal biomass was found to be significantly increased (ca. 1.34-fold increased compared to control infected with CYR23; Figure 6D). The same result was observed after inoculated with CYR31. Quantification of the expansion in uredinia development in leaves was observed to increased 1.19-fold compared with controls treated with BSMV::γ alone (Figure 6E). These results indicate that TaARPC3 is required for defense signaling in wheat during Pst infection.

To further understand the how TaARPC3 participates in wheat resistance, H₂O₂ accumulation was evaluated. As shown in Figure 7, H₂O₂ production in TaARPC3-silenced wheat showed a significant reduction compared with BSMV:γ-treated plants after CYR23 inoculation (Figures 7A,B). To confirm a decrease in ROS accumulation, we next assayed the expression levels of selected genes involved in the ROS signaling pathway [e.g., catalase (TaCAT) and superoxide dismutase (TaSOD)] in TaARPC3-knockdown plants following infection with Pst. As shown in Figure 7C, we observed an increase in the accumulation of TaCAT in TaARPC3-knockdown plants compared with mock-inoculated or BSMV:γ control plants. Conversely, TaSOD transcripts showed no significant change after TaARPC3 silencing.

In addition to the induction of H₂O₂ accumulation, we also observed that pathogen-induced cell death exhibited a similar decrease in the TaARPC3-knowdown plants (Figure 8A), as evaluated by microscopy (Figure 8B). A histological examination of Pst growth was performed in both control and silenced plants by analyzing the length of hyphae and the number of hyphal branches and the formation of haustorial mother cells at infection sites (Supplementary Table S1). In short, we found that the hyphal branch between the BSMV:TaARPC3 and BSMV:γ plants at 24 hpi showed no significant change after inoculation with both CYR23 and CYR 31 (Supplementary Table S1), while the hyphal length was longer in TaARPC3 knock-down plants (Figure 8C and Supplementary Table S1). In addition, the colony area per infection site in the incompatible interaction was obviously larger in TaARPC3 silenced plants compare with the control group at 120 hpi in incompatible interaction (Figure 8D and Supplementary Table S1). In total, these results indicated that a reduction in TaARPC3 expression compromises resistance signaling in response to Pst infection.