Article Figures & Data
Figures
- Figure 1.
HCT1806 and HCT4918 suppressed Rp1-D21-induced HR. A, The positions of genes (HCT1806, HCT4918, AAS6469, and AAS3125) relative to the SNP on chromosome 1 were shown to be highly associated with variation in Rp1-D21-induced HR from a previous study (Olukolu et al., 2014). Arrows indicate the direction of transcription of the genes. B, The phenotypes resulting from transient coexpression of HCT1806, HCT4918, AAS6469, AAS3125, or Gus with Rp1-D21. The pictures were taken at 3 d after inoculation. The two pictures show the same leaf before (left) and after (right) ethanol clearing. C, Ion leakage conductivity was measured at 60 h after coexpression of HCT1806, HCT4918, AAS6469, AAS3125, or Gus with Rp1-D21. Significant differences (P < 0.05) between samples are indicated by different letters (a and b). D, Total protein was extracted from agro-infiltrated leaves at 30 h post inoculation (hpi). Anti-HA was used to detect the expression of Rp1-D21, and anti-GFP was used to detect HCT1806, HCT4918, AAS6469, AAS3125, or Gus. Equal loading of protein samples is shown by Ponceau S staining. Each experiment was performed three times with similar results.
- Figure 2.
Phylogenetic analysis of HCTs from diverse plant species. The two maize HCTs (HCT1806 and HCT4918) colocalized with the SNP associated with Rp1-D21-induced HR are indicated in red boxes, and the other Rp1-D21-induced HCTs used for transient expression in N. benthamiana are indicated in blue boxes. The maize HCTs with expression that is up-regulated or down-regulated by the presence of Rp1-D21 (Supplemental Table S1) are indicated by up or down arrows, respectively. All gene names starting with GRMZM and AC215260.3 are derived from maize. At, Arabidopsis; Bd, B. distachyon; Cc, Coffea canephora; Gm, Glycine max; Hv, barley; Mt, Medicago truncatula; Nt, Nicotiana tabacum; Os, rice; Sb, sorghum (Sorghum bicolor); Sl, Solanum lycopersicum; St, Solanum tuberosum.
- Figure 3.
Investigating the function of maize HCT homologs in Rp1-D21-induced HR. A, The up-regulated HCT homologs were transiently coexpressed with Rp1-D21 into N. benthamiana. The representative leaves were photographed at 3 d after inoculation (upper), and the same leaves were cleared by ethanol (lower). B, Ion leakage conductivity was measured at 60 h after coexpression of Gus or HCT homologs with Rp1-D21. Significant differences (P < 0.05) between samples are indicated by different letters (a–c). C, Total protein was extracted from agro-infiltrated leaves at 30 hpi. Anti-HA was used to detect the expression of Rp1-D21, and anti-GFP was used to detect the expression of Gus or HCT homologs. Equal loading of protein samples is shown by Ponceau S staining. The experiments were repeated three times with similar results.
- Figure 4.
Mutations in the conserved residues required for HCT activity did not affect the suppressive role of HCT on Rp1-D21-induced HR. A, Multiple sequence alignment of plant HCTs. The His residue in the conserved HxxxD motif is boxed. B, The HR phenotypes resulting from transient coexpression of HCT1806, HCT4918, and their mutant derivatives with Rp1-D21. C, Ion leakage conductivity was measured at 60 h after leaf infiltration. Significant differences (P < 0.05) between samples are indicated by different letters (a and b). D, Total protein was extracted from agro-infiltrated leaves at 30 hpi. Anti-HA was used to detect the expression of Rp1-D21, and anti-GFP was used to detect the expression of HCT homologs or Gus. Equal loading of protein samples is shown by Ponceau S staining. The experiments were repeated three times with the same results.
- Figure 5.
Investigating the interactions between Rp1-D21/Rp1-dp2 and HCT1806/HCT4918/HCT7251 by Co-IP assay. EGFP- and 3× HA-tagged constructs were transiently coexpressed in N. benthamiana, and samples were collected at 30 hpi for Co-IP assay. Protein extracts were immunoprecipitated (IP) by anti-GFP (α-GFP) microbeads and detected (immunblotted [IB]) by anti-GFP and anti-HA (α-HA) antibodies. In the rightmost lane of the IP:α-GFP/IB:α-GFP, an arrow indicates the target band. The experiments were repeated three times with the same results.
- Figure 6.
Investigating the interactions between Rp1 proteins and HCT1806/HCT4918/HCT7251 by BiFC assay in N. benthamiana. Rp1-D21/Rp1-D/Rp1-dp2 was fused with cEYFP, and HCT homologs were fused with nEYFP vectors. The cEYFP- and nEYFP-derived constructs were transiently agro-infiltrated into N. benthamiana. The pictures were taken at 40 h postinfiltration under a confocal microscope. Green fluorescence indicates interaction between constructs tagged with nEYFP and constructs tagged with cEYFP. The experiments were repeated three times with the similar results. Bar = 20 µm.
- Figure 7.
Model for the suppression and activation of Rp1 proteins. Rp1 proteins form homo- and heteromers (Wang et al., 2015b). The inhibited state of Rp1 proteins (left) is maintained through autoinhibitory intramolecular interaction and interactions between HCT1806/HCT4918 and the CC domain. In the activated state (right), pathogen effectors modify HCT1806/HCT4918, disrupting the interaction between Rp1 proteins and HCT1806/HCT4918. This, in turn, disrupts the autoinhibitory intramolecular interactions; the interaction between the LRR and NB-ARC domains is strengthened, and the CC/NB-ARC interaction is weakened, leading to activation and HR. Blue and purple triangles indicate ADP and ATP, respectively. Arrows indicate intra- and intermolecular interactions. The putative pathogen effector is labeled with a red star. The rationale behind the models is explained in the text.
Tables
- Table I. Summary of the state of knowledge of the cofactors and interaction domains of plant NLRs
Abbreviations are as follows: Resistance to Pseudomonas syringae pv maculicola1 (RPM1), Resistance to Pseudomonas Syringae2 (RPS2), Suppressor of npr1-1, constitutive1 (SNC1), HopZ-Activated Resistance1 (ZAR1), Mildew A Locus10 (MLA10), Bacterial spot2 (Bs2), Panicle blast1 (Pb1), Constitutive expresser of PR genes1 (CPR1), basic Helix-loop-Helix84 (bHLH84), Enhanced disease susceptibility1 (EDS1), avrPphB susceptible1 (PBS1), HopZ-ETI-deficient1 (ZED1), Squamosa promoter binding protein-like6 (SPL6), suppressor of G2 allele of skp1 (SGT1), Ran GTPase activating protein2 (RanGAP2), and Bsu-like protein1 (BSL1). FL, Full length; TF, transcription factor; XNL, no typical CC or TIR domain was found in the N terminus; TNL, Toll-IL1 receptor-nucleotide binding LRR; —, no data available.
Host Species and Gene Protein Type Pathogen Effector Cofactors Types of Cofactors Interaction Domains with Cofactors Source Arabidopsis RPM1 CNL Pseudomonas syringae AvrRpm1, AvrB RIN4 Unknown CC, FL Mackey et al. (2002) RPS2 CNL P. syringae AvrRpt2 RIN4 Unknown CC, FL Axtell and Staskawicz (2003); Mackey et al. (2003) CPR1 F-box FL Cheng et al. (2011) RPS4 TNL P. syringae AvrRps4 bHLH84 TF FL Xu et al. (2014) EDS1 Lipase-like FL Bhattacharjee et al. (2011); Heidrich et al. (2011) RPS5 CNL Peronospora parasitica AvrPphB PBS1 Kinase CC, FL Ade et al. (2007) RPS6 TNL P. syringae AvrHopA1 EDS1 Lipase-like FL Bhattacharjee et al. (2011) SNC1 TNL P. syringae — bHLH84 TF FL Xu et al. (2014) CPR1 F-box FL Cheng et al. (2011) EDS1 Lipase-like FL Bhattacharjee et al. (2011) ZAR1 CNL P. syringae AvrHopZ1a ZED1 Pseudokinase CC, FL Lewis et al. (2013) Barley MLA10 CNL Blumeria graminis AvrA10 WRKY1, WRKY2 TF CC, FL Shen et al. (2007) MYB6 TF CC, FL Chang et al. (2013) Maize Rp1-D CNL P. sorghi — HCT homologs Lignin biosynthesis FL This study; Wang et al. (2015b) Rp1-dp2 CNL — — HCT homologs Lignin biosynthesis CC, FL This study; Wang et al. (2015b) Rp1-D21 CNL — — HCT homologs Lignin biosynthesis CC, FL This study; Wang et al. (2015b) Nicotiana tabacum N TNL Tobacco mosaic virus P50 SPL6 TF TIR, LRR, FL Padmanabhan et al. (2013) Pepper (Capsicum annuum) Bs2 XNL Xanthomonas campestris pv vesicatoria AvrBs2 SGT1 Molecular cochaperone LRR, FL Leister et al. (2005) Potato (Solanum tuberosum) Rx CNL Potato virus X Coat protein RanGAP2 Small GTPase CC, FL Tameling and Baulcombe (2007) R2 CNL Phytophthora infestans AVR2 BSL1 Phosphatase FL Saunders et al. (2012) Rice Pb1 CNL Magnaporthe oryzae — WRKY45 TF CC, FL Inoue et al. (2013) Pit CNL M. oryzae — Rac1 Small GTPase NB-ARC, FL Kawano et al. (2010) Tomato (Solanum lycopersicum) Prf XNL P. syringae AvrPto, AvrPtoB Pto Kinase CC, FL Mucyn et al. (2006)
Supplemental Data
Supplemental Figures and Tables
Files in this Data Supplement:
- Supplemental Data - Supplemental Tables 1 and 2
- Supplemental Data - Supplemental Figures 1-5
