Plant materials and growth conditions
All the Arabidopsis used in this research were under the background of ecotype Col-0. The PIP2A-RFP74, VHA-a1-mRFP38, mCherry-CLC241, Rha1-mCherry33, 35S::PDLP550, pCTL1::CTL1-GFP49, coi1-275, jazQ76, ctl 49, sld1 sld250, tet855, aos62, patl277, patl352 were described previously. And patl1 (SalK_056190.2) were obtained from the Arabidopsis Biological Resource Center (ABRC).
The seeds of Arabidopsis thaliana, tobacco plants (Nicotiana benthamiana) and cotton plants (Gossypium hirsutum) used in this study were grow in long days (16-h light/8-h dark) under 22℃.
The rice (Oryza sativa) seeds were washed with 75% ethanol for 1 minute, and with 20% (v/v) NaClO for 30 minutes, then rinsed five times with sterile water. The mature embryos were grown on N6D2 medium and cultured in dark at 28℃ for 14 days to induce callus.
Plant Treatment
HARP1 was identified from the oral secretions of cotton bollworm (Helicoverpa armigera)35 and the sequence information can be found in NCBI (https://www.ncbi.nlm.nih.gov/protein/XP_047035071.1?report=genbank&log$=protalign&blast_rank=1&RID=F3YF3FK8016). To detect the effector activity of V-HARP1, the second pair of true leaves at the rapid expanding stage (about 18-day-old seedlings) were wounded (punched 3 ~ 4 holes per leaf) and the unwounded leaves were used as negative control, the V-HARP1 and Venus protein solutions (1 mg/ml) were painted to the wounding sites immediately. Four hours later, leaves were harvested and used for qRT-PCR analyses of the indicated wounding induced gene expressions.
To observe the import of HARP1 in plants, the leaves of Arabidopsis, cotton and tobacco were cut into small pieces with a hole in the center and incubated with V-HARP1 or its truncated forms (1 mg/ml) for 2–4 hours. Samples were then washed with PBS containing 0.08% BSA for at least 5 times, 20 minutes for each time and detected under confocal microscopy. As a negtive control, the Venus (1 mg/ml) were used for incubation with plant leaves like V-HARP1 and followed by the same washing procedure to insure get rid of all the extra proteins adhering to the surface and subsequently for confocal microscopy. Under our experimental condition, Venus along cannot be detected in leaf cells.
For endocytosis inhibition assay, endocytosis inhibitors of Tyrphostin A23 (A23, Sigma-Aldrich), Wortmannin (Wm, Sigma-Aldrich) and Brefeldin A (BFA, TCI) were dissolved in Dimethyl sulfoxide (DMSO, Sigma-Aldrich). The wounded Arabidopsis leaves were incubated with 50 mM Tris-HCl (pH 8.0) containing 30 µM A23, 30 µM 30 Wm and 50 µM BFA, respectively and leaves incubated with 50 mM Tris-HCl (pH 8.0) containing equal amount of DMSO were used as mock treatment. After half an hour, V-HARP1 was added and incubated for another 2–4 hours before confocal microscopy assay.
For the assay of jasmonate effects on V-HARP import, MeJA (Sigma-Aldrich) was dissolved in ethanol and diluted in double-distilled water to a final concentration of 50 µM. As the mock treatment, water solutions with equal volumes of ethanol were used. 18-day-old Arabidopsis were sprayed with water solutions of MeJA and ethanol, respectively. Two hours later, detached leaves were punctured and incubated with the V-HARP1 solutions for 2–4 hours and then observed under confocal microscopy.
Confocal Microscopy
Venus was fused to the N-terminal of HARP1 (V-HARP1) for visualization. After incubation with Venus or V-HARP1 followed by 4–5 times washing, samples were detected by confocal microscopy. To detect HARP1 in cells, regions 100 ~ 600 µm from the wounding site were selected for observation (Supplementary Fig. 18). We found that the fluorescent signal from the Venus treated samples were hardly to detect, therefore, excluding auto fluorescing which might be caused by wounding and the possibility that Venus itself could enter plant cells under our experimental condition. To label plasma membrane and trace the internalized endosomes, samples were incubated with 2 µM FM4-64 (Invitrogen) in the dark for 5–7 minutes, and washed twice with double-distilled water.
All images and movies were taken by confocal systems, Leica SP8 or Olympus spinSR equipped with different immersive objectives (Leica SP8 with 20XW/NA 0.75 and 60XW/NA 1.2, Olympus spinSR with 30XSil/NA 1.05 and 60XSil/NA 1.30). The confocal z-axis resolution range is about 1240 ~ 647 nm. The moving HARP1 granules were traced by time series at a fixed layer. For multi-channel images, we use sequential imaging procedure to exclude fluorescence signals crosstalk.
The excitation/emission wave lengths for YFP and FM4-64/mRFP/mCherry signals were 515/525–560 and 561/610–650 nm, respectively. And Images were captured with strictly identical acquisition parameters for the quantitative fluorescence intensity. The fluorescence signal intensity was calculated using ImageJ software and statistically analyzed with GraphPad Prism software.
Immuno-localization
After the Arabidopsis leaves were incubated with Venus or V-HARP1 followed by 4–5 times washing, samples were cut into pieces (1–2 mm2) and immediately fixed in 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS) at 4℃ overnight. Fixed leaves were embedded in LR White resin (Sigma-Aldrich) after dehydration through a graded alcohol series. Sections were prepared on a Leica Microsystem UC7 ultramicrotome. Ultrathin sections of approximately 70 nm were mounted onto nickel grids for immuno-gold labeling.
Immuno-gold labeling was performed as follows: 3% acetylated bovine serum albumin C in PBST (PBS + 0.05% Tween 20) (BSA/PBST) for 1 hour; anti-GFP antibody (1:50 dilution in 3% BSA/PBST) for 4 hours; PBST sixteen times for 2 min; goat anti-mouse IgG conjugated with 10-nm colloidal gold particles (1:50 diluted in 3% BSA/PBST, Sigma-Aldrich) for 2 hours; PBST ten times for 2 min; and water twice for 2 min. Grids were observed under 120 kV transmission electron microscope (TEM) after staining. The replicate sections of Venus treated samples had no signals of immuno-gold in cells indicating that the signal is specific and Venus alone could not enter plant cells.
Prokaryotic Expression And Purification Of Tested Proteins
For His-fused proteins (Venus, V-HARP1, V-HARP1δC5, V-HARP1δN34, V-HARP1δN39, V-HARP1δN44, V-HARP1δN49, dsRed-HARP1, mCherry-HARP1), the ORF of the indicated genes were inserted into pETDuet-1 vector with a His N-terminal fusion. For GST-fused proteins (GST-CTL1EC1, GST-PATL2C110, GST-TET8EC2), the ORF of the indicated genes were inserted into pGEX-4T-1 vector with a GST N-terminal fusion. The primers used for the vector constructions are given in Supplementary Table 1.
The constructed vectors were transformed into Escherichia coli strain BL21 (DE3) strain. 0.25 mM isopropyl β-D-thiogalactopyranoside (IPTG) was used to induce protein expression. E. coli cells carrying the designed vectors were cultured in 37℃. After IPTG addition, the cells were transferred to 16℃ for another 12–16 hours before harvested. The His fusion proteins were purified by Ni affinity column (Ni-NTA resin, Qiagen) and the GST fusion proteins were purified by Glutathione Sepharose 4B resin (GE Healthcare). The eluted proteins were then concentrated and desalted using an Amicon Ultra-15 Centrifugal Filter Unit (10,000 molecular weight cutoff [MWCO], EMD Millipore) with 50 mM Tris–HCl (pH = 8.0) to a final protein concentration of 1 mg/ml.
Transient expression assay in Nicotiana benthamiana (N. benthamiana)
About 4–5 monoclones of Agrobacterium carrying the target genes were grown together overnight in liquid LB medium under 30 ℃, then were transferred to the new liquid LB medium at a ratio of 1:500 and grown overnight. The cultures were centrifuged at room temperature and cells were resuspended with infiltration buffer (10 mM MgCl2, 10 mM MES, 150 µM Acetosyringone, pH = 5.7) at an OD600 of about 1.0. The cell solutions were infiltrated into the back of tobacco leaves. 2–3 days later, leaves were harvested for BiLC and pull-down assay.
Bimolecular Luciferase Complementary (BiLC) Assay
The BiLC assays were performed as described78. For HARP1 interaction with CTL1 and PATL2, CTL1 and PATL2 were fused to the amino-terminal half of LUCIFERASE (nLUC), HARP1 was fused to the carboxyl-terminal half of LUC (cLUC). For HARP1 interaction with TET8, HARP1 was fused to nLUC and TET8 was fused to cLUC. cLUC and nLUC alone were used as controls. The oligonucleotide primers used for these vectors are given in Supplementary Table 1. Agrobacterium cell solutions were infiltrated into tobacco leaves. Luciferin (1 mM) was infiltrated before LUC activity was monitored after 2 d.
GST Pull-down Assay And Immunoblot
Full-length V-HARP1 were expressed in N. benthamiana leaves. V-HARP1 and Venus were inserted into pCAMBIA1300 vector with a flag C-terminal fusion and then transiently expressed in N. benthamiana. The soluble proteins of N. benthamiana leaves transiently expressing V-HARP1-flag and Venus-flag (control) were extracted in extraction buffer (50 mM HEPES pH = 7.5, 50 mM NaCl, 10 mM EDTA, 10% glycerol, 0.2% Triton X-100, 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 50 µM MG 132, and 500 × protease inhibitor cocktail). His-fused V-HARP1δN39, V-HARP1δN44 were prokaryotically expressed and purified. About 10 µg proteins of prokaryotically expressed GST, GST-CTL1EC1, GST-PATL2C110 and GST-TET8EC2 were added 20 µL Glutathione Sepharose 4B resins respectively, and then incubated with total protein extracts (about 250 µg) of N. benthamiana leaves in 1 mL extraction buffer or His-fused V-HARP1δN39, V-HARP1δN44 (about 10 µg) in 1 mL 50 mM Tris–HCl (pH = 8.0) at 4℃ with rotation. Then, the resins were washed with wash buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 10 mM EDTA, 10% glycerol, 0.1% Triton X-100) for several times. Anti-Flag antibody (ABclonal; dilution, 1:2,500) was used to detect V-HARP1-flag and Venus-flag or anti-His antibody (ABclonal; dilution, 1:2,500) was used to detect V-HARP1δN39 and V-HARP1δN44 in input and output. Anti-GST (ABclonal; dilution, 1:2,500) antibody was used to detect GST, GST-CTL1EC1, GST-PATL2C110 and GST-TET8EC2 in input.
Yeast Two Hybrid
V-HARP1 variants were introduced into the pGBKT7 (Clontech). CTL1EC1, PATL2C110 and TET8EC2 were introduced into the pGADT7 (Clontech). The oligonucleotide primers used for these vectors are given in Supplementary Table 1. A LiCl polyethylene glycol method was used to transfer the indicated plasmids into yeast strain AH109 (Clontech). Transformants were grown on SD-Leu-Trp mediums for 2–3 days and then tested on SD-Leu-Trp-His mediums (-L-T-H) or SD-Ade-Leu-Trp-His mediums (-A- L-T-H) with the indicated 3-amino-1,2,4 triazole (3-AT). At least 10 individual clones for each transformant were analyzed to confirm the interactions.
Insect Feeding Test
The cotton bollworm (Helicoverpa armigera) larvae were obtained from the Institute of Zoology, Chinese Academy of Science. About 30 synchronous third instar larvae were fed on 60 Arabidopsis plants of 20 days old. After fed on for 3–4 days, weight increases were recorded.
RNA-seq And Transcriptome Analysis
The leaves of 10-day-old wild type (Col-0) and JA synthesis mutant (aos) were wounded (W) and harvested 2 hours post wounding. The untreated plant leaves (CK) were used as control. Total RNA was extracted using the Plant RNA purification kit (Qiagen, 74904). Library construction and RNA-sequencing with three biological replicates was performed on an Illumina HiSeqXten platform (Illumina, San Diego, CA, United States) at Majorbio (Shanghai, China). The clean reads were mapped to the Arabidopsis genome (TAIR10) using Hisat2 v2.0.4. HTSeq v0.9.1 to count the reads numbers mapped to each gene. Fragments Per Kilobase of exon model per Million mapped fragments (FPKM) was calculated based on the gene length and the mapped-reads counts. Genes with a fold-change greater than 2 and FDR less than 0.05 were considered as differentially expressed genes (DEGs) using the R package DESeq279. Gene Ontology (GO) enrichment and network analysis were performed by using the Cytoscape v3.8.2 plug-in ClueGO80. Significant enrichment was defined as FDR less than 0.05. GO clusters were inferred by Kappa score. The complete list can be found in Supplementary Table 2. The raw data are deposited in the NCBI (BioProject accession number: PRJNA760932) and will be released as soon as publication. The reviewer link: https://dataview.ncbi.nlm.nih.gov/object/PRJNA760932?reviewer=kbk81o7uvur0kpn4dh4kpus2lr.
Gene Expression Analyses
Plant total RNA was extracted by Trizol reagent (Invitrogen). 1.5 ug of total RNAs were treated with DNase I (1 unit per ul; Fermentas) and used for cDNA synthesis with oligo (dT) primer (TransGen Biotech). qRT-PCR was performed using SYBR green PCR master mix (TaKaRa) on a real-time PCR system (CFX thermocycler; Bio-Rad, Hercules, CA). S18 in Arabidopsis (At4g09800) was used as an internal standard. The gene average expression levels were calculated from 2−ΔΔCt values. At least three biological triplicates with technical triplicates were performed. The oligonucleotide primers for all the genes tested are given in Supplementary Table 1.
Statistical analysis
Data are presented as means ± SEM. Significances were examined by Student’s t test or by one-way or two-way ANOVA followed by multiple comparison tests with GraphPad Prism software. No less than three independent experiments were performed for each assay, and every experiment contains at least three biological replicates.