Genome sequencing and assembly
Commercial DNA extraction kit (Tiangen Biotech, Beijing, China) was used to extract genomic DNA from Verticillium dahliae isolates V991 and 1cd3-2 for long read sequencing. Twenty kilobase-long-insert libraries were sequenced with PacBio RS II SMRT cells. Canu was used to assemble long reads for genome assembly37. Illumina sequencing of 500-bp-insert libraries was used to refine the initial assembly genomes.
Species-specific libraries were used for annotating repeats for each isolate. LTR_FINDER (version 1.05), MITE-Hunter (20100819), Repeat Scout (version 1.0.5) and PILER-DF (version 2.4) were utilized to generate repeat libraries based on a structure-based method and de novo prediction38. PASTE Classifier (version 1.0) was applied for repeat classification and then merged with all repeats from Repbase Database (version 19.06)39. Finally, Repeat Masker was used to predict fungal isolate repeats40.
Gene prediction and annotation
EVM was used to integrate all predicted results41. For de novo prediction, several software programs were used, including Augustus, GlimmerHMM, and SNAP (version 2006-07-28), to scan the repeat-masked genome42. For the homolog-based approach, GeMoMa was used based on Verticillium dahliae VdLs.17, Verticillium alfalfae VaMs.102, Fusarium oxysporum f. sp. lycopersici 4287, Fusarium verticillioides 7600, and Fusarium graminearum PH-143. PASA was used to predict genes based on Illumina sequencing RNA data44.
DNA methylation modification identification
Based on PacBio-generated raw data, 6 mA and 4mC base modifications were identified using PacBio SMRT Analysis 2.3.0 with default settings (https://www.pacb.com/documentation/smrt-analysis-software-installation-v2-3-0/).
Isolate specific genes identification
Based on bidirectional BLAST results, homologous gene pairs (smaller than E-value 1e-20) were retained as core gene sets. Extra gene sets were defined as specific genes to represent the functional specificity of one genome. PCR primers were separately designed based on the gene upstream 1 Kb and downstream 1 Kb to cover gene regions (Supplementary Data 5).
Present/absent variation (PAV) identification
The program Nucmer in Mummer was used to compare two genomes, and then “show-diff” was utilized to identify PAVs. Candidate PAVs were filtered by abandoning sequences homologous with the other genome (threshold: E < 1e-5, coverage>50% and identity>90%). To ensure accuracy, candidate PAVs were aligned with the Pacbio-sequenced raw reads of the other genome and deleted candidates with similar thresholds (E < 1e-5, coverage>60% and identity>90%). Finally, unaligned regions were regarded as lineage-specific regions.
Pathogen cultivation, infection and disease assay
V. dahliae was inoculated on a potato dextrose agar (PDA) plate and placed in an incubator at 25 °C for four days. Fungal colonies were transferred into Czapek medium on a shaker at 120 rpm at 25 °C for four days until the spore concentration reached ~108 spores mL−1. Cotton (Gossypium hirsutum cv. Jin668) was watered with Hoagland solution for 3 weeks under greenhouse conditions of 25 °C to 28 °C under long-days with an 8 h/16 h dark/light photoperiod and a relative humidity of 60%. Three-week-old cotton seedlings were inoculated for two minutes with 1 × 106 conidia mL−1 by the root-dip method using a spore suspension, and the inoculated plants were placed in pots (10 × 10 cm) filled with standard soil mix (Xingyuxing, Wuhan, China). The disease index was scored using at least 25 plants per treatment and repeated at least three times45. The disease grade was classified as follows: 0 (no symptoms), 1 (0%–25% wilted leaves), 2 (25%–50% wilted leaves), 3 (50%–75% wilted leaves) and 4 (75%–100% wilted leaves).
Generation of gene deletion mutants and mutant complementation
Performing A. tumefaciens-mediated V. dahliae transformation and PCR-based transformants screening to generate the knockout mutants28. To produce complementary transformants, the coding sequence for SP3 with its native promoter was cloned and inserted into the binary vector p823-GFP encoding resistance to G418, and SP3 was reintroduced into the ΔSP3 strains. Complemented transformants were obtained using an A. tumefaciens -mediated transformation method28. All primers used are shown in Supplementary Data 5.
Yeast signal sequence trap system
The predicted signal peptide of SP3 was cloned and inserted into pSUC2T7M13ORI (pSUC2), and the resulting plasmid was transformed into Yeast YTK12 strains. All strains were screened on CMD-W medium (0.67% yeast N base without amino acids, Trp dropout supplement, 2% sucrose, 0.1% glucose, and 2% agar), and positive transformants were incubated on YPRAA medium (1% yeast extract, 2% peptone, 2% raffinose, 2% agar, pH 5.8)46. Invertase enzymatic activity was assessed by a 2,3,5-triphenyltetrazolium chloride (TTC) reduction assay47. Total yeast cells were collected and washed with double distilled water. The pellet was resuspended in 0.1% colorless TTC dye and incubated at 30 °C for 1-2 h, and the color change was determined at room temperature. The empty pSUC2 and pSUC2-Avr1bSP vectors were used as negative and positive controls, respectively.
Total RNA extraction and RT‒qPCR analysis
Total RNA was extracted using an RNA Extraction Kit (Tiangen Biotech, Beijing, China). First-strand cDNA was generated from 3 μg of total RNA using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and diluted 100 times with double distilled water. Reverse transcription quantitative PCR was performed using a 7500 Real Time PCR system (ABI, Foster City, CA, USA) in a 15 μL reaction volume. qRT‒PCR was performed as follows: an initial 95 °C denaturation step for 3 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 45 s. The housekeeping genes GhUB7 and v991_EVM0005718 were used as internal controls for cotton and V. dahliae, respectively. The primers used in this study are listed in Supplementary Data 5.
Sample preparation and RNA-seq
We conducted bidirectional transcriptome analysis on hypocotyls of cotton during the whole period of interaction (0, 3, 6, 9, 12, 15, 18, 21 dpi). RNA was isolated from hypocotyls of cotton infected with V. dahliae. The library preparations were sequenced using the Illumina NovaSeq 6000 platform, and 150-bp paired-end reads were generated. Sequencing adapters were removed, and consecutive low-quality bases were trimmed from both the 5’ and 3’ ends of the reads. High-quality RNA-Seq reads were aligned to the V. dahliae genome using HISAT2 with default parameters. Transcripts of each sample were assembled using Stringtie, and pairwise comparisons between samples were performed using the edgeR package48. We used FPKM > 0.01, fold change≥2 and P < 0.05 as thresholds to screen for DEGs. The RNA-Seq reads were also processed and mapped to cotton in the same way. We used FPKM > 1, fold change≥2 and P < 0.05 as the thresholds to screen for DEGs in the cotton transcriptomes. The threshold P < 0.05 was selected to identify significantly enriched GO terms.
Synthesis and characterization of polyethyleneimine-coated MXene quantum dots
Polyethyleneimine-coated MXene quantum dots (PEI-MQDs) were synthesized from Ti3C2 MXene powder (Jilin Science and Technology) and 25 mg of polyethyleneimine (PEI) (Sigma, MV 1800). Briefly, the mixture of Ti3C2 MXene powder and PEI was added to 10 mL DI water and then perfused with N2 for 5 min The solution was then incubated at 120 °C for 8 h in a drying oven. The pH of the obtained solution was adjusted to approx. 7.0, and the final solution was dialyzed for 24 h. After dialysis, the solution was freeze-dried for 24 h, and the final products were stored in a refrigerator until further use. For nanoparticle characterization, transmission electron microscopy (TEM) images were captured by an H-7650 (HITACHI, Japan). The surface charge of PEI-MQDs was measured by using a zetasizer (NanoBrook 90Plus Zeta). The ζ-potential characterization (NanoBrook 90Plus Zeta) confirmed the presence of a negative charge for PEI-MQDs.
Catalytic activity of PEI-MQDs
The catalytic activity of PEI-MQDs was evaluated by measuring their peroxidase-mimicking activities with the use of the colorimetric substrate 3,3,5,5-tetramethylbenzidine (TMB), which is oxidized in the presence of H2O2 to produce a color reaction49.
Root absorption and FITC-PEI-MQDs application to cotton plants
Cotton roots were incubated in a solution containing PEI-MQDs (50 mg/L), with the root completely submerged in the dark for 3 h. The PEI-MQDs solution was removed, and the roots were placed in water for one day before inoculation with V991. Two treatment replicates were divided into two groups: each group had at least 16 plants, one group treated with FITC-PEI-MQDs and another with water as a control. The mixture was composed of 200 μL anhydrous ethanol, 50 FITC (2.5 mg/mL), 1 mL 50% alcohol and 2 mg/mL PEI-MQDs, and kept in a 20 mL glass vial at 1000 × g for 10 min in the dark. The resulting mixture was purified using a 10-kDa filter (4200 × g for 5 min at least 8 times) to remove the dissociated chemicals. The final solution was labelled with FITC-PEI-MQDs and stored at 4 °C until use.
ROS determination and enzyme activity assay
DCF, DHE and HPF fluorescent dyes are used to visualize ROS in leaf tissues50. Leaf discs (diameter, 5 mm) were incubated with either 25 μM DCF for 30 min or 10 μM DHE dye for 30 min or 10 μM HPF for 1 h in darkness (in TES infiltration buffer, pH 7.5). After incubation, the leaf discs were rinsed with deionized water three times and mounted onto a glass slide. A coverslip was placed on the top to seal the well while ensuring that no air bubbles remained trapped. The samples were imaged using a Leica SP8 confocal microscope (Leica Microsystems, Germany). Six repetitions were performed. The fluorescence intensities of DHE, DCF and HPF were calculated by LAS (Leica Application Suite) AF Lite software.
Hydrogen peroxide (H2O2), malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GSH-PX) and peroxidase (POD) were analyzed using H2O2, MDA, CAT, GSH-PX and POD assay kits (Suzhou Grace Biotechnology Co., Ltd.).
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