Polyethyleneimine-coated MXene quantum dots improve cotton tolerance to *Verticillium dahliae* by maintaining ROS homeostasis

Verticillium dahliae infects over 400 plant species, including cotton, causing severe yield losses. Cotton-infecting strains are categorized into defoliating and nondefoliating pathotypes, with defoliating strains causing rapid leaf necrosis and plant death. Understanding genomic variation that underpins pathogenicity is important for disease control. Pathogen virulence evolution is often driven by genomic structural variation and lineage-specific regions enriched for secreted proteins and effectors that modulate host defenses. Prior work indicates that defoliating isolates can activate ethylene signaling in cotton to enhance virulence, and that metabolic reprogramming and reactive oxygen species (ROS) are central in plant-pathogen interactions. However, the role of ROS homeostasis in cotton–V. dahliae interactions, particularly with defoliating isolates, remains unclear. This study investigates genome-level differences between a defoliating (V991) and a nondefoliating (1cd3-2) isolate, defines temporal stages of infection through co-transcriptomics, identifies virulence determinants such as the secreted protein SP3, and evaluates a nanomaterial-based strategy (PEI-MQDs) to modulate ROS and improve cotton tolerance.

Previous studies have shown that lineage-specific genomic regions in V. dahliae harbor effectors and secreted proteins affecting host immunity. Defoliating isolates can promote ethylene biosynthesis in cotton via specific effectors, and attenuation of ethylene signaling enhances resistance. In plants, phenylpropanoid metabolism reprograms during stress, influencing lignin and flavonoid biosynthesis; certain cotton genes that redirect this flux increase tolerance to V. dahliae and herbivory. ROS bursts are hallmark defenses, but excessive ROS can lead to necrosis and susceptibility; in cotton, perturbations (e.g., CYP82D/GhSSN knockdown) can trigger lethal ROS accumulation. Quercetinases such as VdQase in V. dahliae catabolize flavonols, potentially depleting antioxidant pools. Nanomaterials, including cerium oxide and zinc oxide-based systems, can mimic antioxidant enzymes to scavenge ROS and enhance plant stress resistance, motivating exploration of PEI-MQDs in disease management.

Two Verticillium dahliae isolates were selected: defoliating V991 and nondefoliating 1cd3-2. High-quality reference genomes (8 chromosomes) were assembled using PacBio RS II long reads (20 kb inserts) assembled with Canu, and polished with Illumina 150-bp paired-end reads. Repeats were annotated using LTR_FINDER, MITE-Hunter, RepeatScout, PILER-DF, PASTEC, and RepeatMasker. Gene prediction integrated de novo (Augustus, GlimmerHMM, SNAP), homology-based (GeMoMa with related fungi), and transcript-based (PASA) evidence via EvidenceModeler. DNA base modifications (6mA, 4mC) were called from PacBio data. Core and isolate-specific genes were defined by bidirectional BLAST; presence/absence variations (PAVs) were identified with MUMmer (nucmer, show-diff) and filtering against the counterpart genome and raw reads. Cotton–fungus co-transcriptome time-course profiling was performed on infected cotton hypocotyls at 0, 3, 6, 9, 12, 15, 18, and 21 dpi. RNA-seq libraries (NovaSeq 6000) were mapped with HISAT2, assembled with StringTie, and DEGs identified with edgeR (fungus: FPKM>0.01, FC≥2, P<0.05; cotton: FPKM>1, FC≥2, P<0.05). GO enrichment used Fisher’s exact test. Stage-specific clustering used fuzzy c-means. A secreted protein gene, SP3, identified in a V991 PAV, had its signal peptide validated via a yeast pSUC2 secretion trap assay. SP3 knock-out mutants (ΔSP3-1, ΔSP3-2) and complementation lines (Comp-1, Comp-2) were generated by Agrobacterium tumefaciens-mediated transformation; transformants were PCR-verified and phenotyped for growth and virulence in cotton. Cotton infection assays used Gossypium hirsutum cv. Jin668 seedlings inoculated by root-dip with 1×10^6 conidia mL^-1; disease index scoring used a 0–4 scale. Fungal biomass was quantified by qPCR (ITS target, GhUB7 reference), fungal recovery assays, and vascular browning observations in stem sections. ROS dynamics in leaves were imaged by confocal microscopy using DCF (H2O2), DHE (O2−), and HPF (•OH) dyes; fluorescence intensities were quantified across timepoints and treatments. Polyethyleneimine-coated MXene (Ti3C2) quantum dots (PEI-MQDs) were synthesized by hydrothermal treatment of Ti3C2 MXene with PEI in water (120 °C, 8 h), followed by pH adjustment, dialysis, and lyophilization. Nanoparticles were characterized by TEM (size, d-spacing), zeta potential, and fluorescence spectroscopy; peroxidase-like activity was assayed via TMB/H2O2 colorimetry. In vitro ROS-scavenging efficiencies for O2−, H2O2, •OH, and ONOO− were measured at 50 mg/L PEI-MQDs. Effects on fungal radial growth on PDA were tested. Uptake into cotton roots was assessed by FITC-labeled PEI-MQDs and confocal imaging (membrane colocalization). In planta effects of PEI-MQDs were tested by pretreating roots with 50 mg/L PEI-MQDs (3 h), followed by inoculation with V991. Disease severity, fungal biomass, MDA and H2O2 contents, and activities of CAT, GSH-Px, and POD were quantified at specified timepoints. Statistical analyses included Student’s t tests with significance thresholds as indicated.

• Genome assemblies of V991 and 1cd3-2 contained 10,941 and 10,971 protein-coding genes, respectively, with isolate-specific genes enriched in PAV regions. PAVs comprise 5.2%–8.1% of the genome yet harbor 33.9%–41.7% of specific genes; 227 V991-specific genes (15 predicted secreted proteins) and 193 1cd3-2-specific genes (16 predicted secreted proteins) were identified. A 106-kb insertion on V991 chr5 included 20 specific genes, 3 encoding secreted proteins (SP3, SP5, SP8). • Co-transcriptomics distinguished two infection stages: Stage I (3–9 dpi) and Stage II (12–21 dpi) for both fungus and host. In V991, Stage II showed upregulation of virulence-related categories (effectors, P450s, transporters, CAZy, PHI genes), with predicted secreted proteins in PAVs predominantly expressed at Stage II. • Cotton responses at Stage I were enriched in cell wall metabolism and xylem/phloem histogenesis, while Stage II was enriched in defense, stress responses, and response to ROS; more PR, PCD, and ROS-related genes were induced with V991 than with 1cd3-2. • ROS imaging revealed low ROS at early timepoints; ROS accumulation began at 9 dpi and rose markedly by Stage II in V991-infected leaves, correlating with higher fungal biomass and severe symptoms. ROS levels were significantly lower with 1cd3-2 infection. • SP3, a V991-specific secreted protein with a functional signal peptide and predicted quercetinase, was highly expressed in planta from 9–21 dpi (peak at 15 dpi). ΔSP3 mutants showed normal growth but significantly reduced virulence, fewer chlorotic leaves, and lower disease index than WT and complemented strains, and induced less ROS in infected cotton. • PEI-MQDs characterization: average diameter 3.85 ± 1.5 nm; d-spacing 0.229 nm; zeta potential +31.71 ± 2.14 mV; emission peak 475 nm (Ex 340 nm). In vitro ROS scavenging at 50 mg/L: O2− 11.70%, H2O2 6.56%, •OH 42.93%, ONOO− 27.23%; retained H2O2 scavenging after exposure, and exhibited peroxidase-like activity. PEI-MQDs did not inhibit V991 radial growth on PDA. • Cotton pretreated with 50 mg/L PEI-MQDs exhibited reduced disease symptoms and disease index, lower fungal recovery and vascular browning, and reduced fungal DNA relative to controls upon V991 challenge. Biochemical assays at 12 dpi showed significantly lower MDA and H2O2 contents and higher activities of CAT, GSH-Px, and POD in PEI-MQDs-treated plants. Confocal assays showed that PEI-MQDs reduced excessive ROS at late infection stage without elevating early-stage ROS.

The study demonstrates that virulence differences between defoliating and nondefoliating V. dahliae isolates are associated with presence/absence genomic variation enriched in secreted proteins and effectors, which are predominantly activated at a late necrotrophic phase (Stage II). In cotton, this phase coincides with strong defense gene induction but also with excessive ROS accumulation that correlates with chlorosis, necrosis, and defoliation, indicating that ROS overaccumulation is a susceptibility factor exploited by highly virulent isolates. Identification of SP3, a V991-specific secreted protein predicted to be a quercetinase, links pathogen virulence to potential depletion of antioxidant flavonoids, thereby facilitating ROS buildup and disease progression. SP3 deletion attenuated virulence and reduced host ROS, underscoring its role in promoting a pathogenic ROS environment. The nanobiotechnology approach using PEI-MQDs effectively maintained ROS homeostasis in infected plants, lowering oxidative damage markers and enhancing antioxidant enzyme activities, which translated into reduced disease severity and fungal biomass without direct antifungal effects. These findings support a model in which defoliating isolates induce and benefit from host ROS, and targeted ROS modulation can mitigate disease outcomes, offering a complementary management strategy to genetic resistance.

This work uncovers a two-stage infection program in cotton–V. dahliae interactions and establishes that late-stage activation of virulence factors, including the V991-specific secreted protein SP3, contributes to excessive host ROS accumulation and severe disease. By synthesizing and applying PEI-MQDs with ROS-scavenging and peroxidase-mimicking activities, the study demonstrates an effective means to maintain ROS homeostasis in cotton, reduce oxidative damage, and improve tolerance to V. dahliae without suppressing fungal growth directly. The results provide mechanistic insight into pathogen-driven manipulation of host ROS and present a nanobiotechnology-based approach to enhance disease management in cotton. Future research should elucidate the regulatory mechanisms governing the transition between biotrophic and necrotrophic stages, define the molecular targets and substrates of SP3 and co-expressed effectors, and assess the efficacy and safety of PEI-MQDs in field conditions and across cultivars.

The molecular mechanisms controlling the transition from Stage I to Stage II remain unresolved. The coordination and regulation of multiple late-stage effectors, including how they co-express to drive ROS accumulation, require further investigation. While PEI-MQDs improved tolerance under controlled conditions, field validations, long-term environmental impacts, and potential effects across diverse cotton genotypes were not addressed. The precise biochemical activity and in planta substrates of SP3 were inferred by prediction and phenotype but not fully characterized biochemically within cotton tissues.