A single-cell leaf transcriptomic atlas of Vitis vinifera during early E. necator infection revealed cell-type-specific temporal dynamics of defense-related gene expression, with epidermal cells showing delayed transcriptional activation relative to other cell types.
Abstract
The powdery mildew pathogen Erysiphe necator poses a major threat to global viticulture, yet the earliest hours of colonization remain transcriptionally uncharacterized at the single-cell resolution. Here, we constructed a single-cell leaf transcriptomic atlas of Vitis vinifera during early E. necator infection by profiling 113,346 cells across five leaf cell types at 0-, 3-, 6-, and 12 h post-inoculation. This revealed cell-type-specific temporal dynamics of defense-related gene expression, with epidermal cells showing delayed transcriptional activation relative to other cell types. Pseudotime trajectory analysis identified four sequential transcriptional states in epidermal cells, and co-expression network analysis uncovered defense-associated gene modules. We identified 230 NLR immune receptor genes exhibiting distinct cell-type-specific expression patterns and 20 small secreted peptide (SSP)-encoding genes differentially expressed in epidermal cells, including four candidates upregulated at 3 or 6 hpi. These findings provide a transcriptomic framework for understanding cell-type-specific defense dynamics and prioritizing candidate genes for functional studies, thereby offering a molecular resource for breeding powdery mildew-resistant grapevine cultivars.
Cercospora leaf spot (CLS), caused by the hemibiotrophic fungus
Cercospora beticola
, is a destructive foliar disease that threatens sugar beet production worldwide. Despite its economic significance, the molecular interaction between sugar beet and
C. beticola
is poorly understood.
We report integrated time-resolved transcriptomics and hormone profiling of CLS infection spanning the asymptomatic and symptomatic phases. Our data revealed broad transcriptional changes, with defense and hormone-related processes being most altered. We detected transcriptional signatures consistent with activation of pathogen-associated molecular pattern- and effector-triggered immunity, along with differential expression of genes encoding putative receptor-like kinases and receptor-like proteins as well as nucleotide-binding (NB) leucine‑rich repeat (LRR) receptors (NLRs). Defense activation was evident as early as 3 days post-inoculation (dpi), including upregulation of pathogenesis-related proteins, genes related to systemic acquired resistance, and the hypersensitive response (HR). At 7 dpi, several biological processes, including defense response, showed overall downregulation, suggesting a pathogen-mediated suppression of transcriptional responses. During the symptomatic phase, defense-related genes—particularly those associated with HR and numerous putative NLRs—were induced again, illustrating a bi-phasic regulation pattern. Cis-element enrichment analysis showed significant overrepresentation of defense-related WRKY and AT-hook motif nuclear-localized protein binding motifs within the protomers of genes upregulated from 11 to 25 dpi. Parallel quantification of major phytohormones demonstrated stage-dependent modulation of infection-related hormone groups across the infection cycle.
Our analysis revealed that a susceptible sugar beet genotype mounts an early and transient defense response against
C. beticola
, which appears to be pathogen-suppressed but later reactivated during symptom development. By integrating time-resolved transcriptome analysis and hormone profiling, our study links major defense transcriptional programs and phytohormone dynamics, providing a comprehensive view of sugar beet responses to CLS that complements and extends previous genotype-focused work.
Leonard Barnabas Ebinezer, Mari Natwick, Lorena I. Rangel et al.· BMC Plant Biology· 0 citations
This study provided valuable insights into the heat stress response mechanisms of alfalfa at the single-cell level by integrating snRNA-seq, WGBS, and pseudo-time trajectory analyses, and demonstrated that MsHSP90 and MsHSFA5 were core transcription factors appearing repeatedly.
Yanmin Hu, Jinsong Zhang, Yao Liu et al.· Plant Physiology· 0 citations
The first spatiotemporal atlas of RKN-infected tomato roots is established and genes associated with giant cell formation are identified, laying a foundation for further research on the establishment of RKN feeding sites, providing novel insights into RKN pathogenic mechanisms, and potentially guiding novel control strategies.
Lijun Jiang, Dan Chen, Qianqian Shi et al.· Molecular Horticulture· 0 citations
Introduction Widespread dicamba use poses challenges of resistance and phytotoxicity. To investigate the temporal molecular response mechanisms of tobacco, this study performed a time-resolved transcriptomic analysis of tobacco seedlings exposed to dicamba. Methods Samples were collected at 6, 24, and 72 h after treatment. Results Results indicated that tobacco exhibits a putative three-phase transcriptional adaptation pattern of “perception-defense-repair”. In the early stage (6h), NtIAA genes were rapidly induced alongside activation of the glutathione system, potentially alleviating oxidative stress; at the mid-stage (24h), enhanced carotenoid synthesis and thylakoid reconstruction appeared to protect photosynthetic structures; at the late stage (72h), the transcriptional response shifted toward systemic repair through secondary metabolism, including phenylpropanoid biosynthesis. Predictive regulatory network analysis suggested that the ERF transcription factor Nitab4.5_0000015g0020 may act as a candidate hub, potentially linking auxin signaling and ribosomal protein genes. Discussion Taken together, this study provides transcriptomic evidence that the NtIAA family may serve as candidate genes in response to dicamba, offering potential genetic candidates for breeding herbicide-resistant crops.
Zhaopeng Luo, Lifeng Jin, Peilin Li et al.· Frontiers in Plant Science· 0 citations
Protoplast isolation is widely used for plant functional genomics and single-cell analyses, but its impact on transcriptional and cell state dynamics remains incompletely understood. Here, we generated time-course RNA-seq data from leaf protoplasts of Arabidopsis, maize, and poplar, sampling at multiple time points following isolation, to systematically characterize global transcriptional dynamics across species. We identified two major drivers of transcriptional variation: a persistent protoplast isolation effect and a progressive time-dependent transcriptional program, which can be divided into early, middle, and late stages corresponding to an immediate stress response, metabolic and chromatin regulation dynamics, and sustained metabolic and proteostasis regulation, together with species-specific differences across stages. We observed a rapid loss of cell-type-specific transcriptional signatures within 6 hours in Arabidopsis and maize, whereas poplar showed a slower decline. Single-nucleus RNA-seq at 6 hours in maize confirmed attenuation of cell-type-specific transcriptional structure. Furthermore, leveraging this time-course dataset enables the identification of aberrant cell states in single-cell RNA-seq data, exemplified by clusters showing elevated activity of protoplast isolation-associated, middle-, and late-stage transcriptional programs characteristic of stress-like states. Together, our results provide a cross-species framework for dissecting protoplast-induced transcriptional and cell state dynamics and facilitate the systematic identification of stress-associated cell states in single-cell transcriptomic data.
Plant development and stress responses are coordinated through phytohormone-mediated gene networks, yet resolving their spatiotemporal crosstalk remains challenging. Here, we generate a single-nucleus transcriptomic atlas of Arabidopsis seedlings, capturing early (0.5 h, 3 h) responses to 9 hormones across ~500,000 nuclei, including auxin, cytokinin, ABA, gibberellin, strigolactone, brassinosteroid, ethylene, JA, and SA. Within this window, most hormones showed rapid and cell-type-specific responses, whereas JA, SA, ABA showed more sustained and convergent responses by 3 h. Co-directional transcriptomic overlap was strongest at 0.5 h, while the JA-, SA-, and ABA-related responses showed the highest overlap at 3 h. Pathway-level analysis indicated asymmetric relationships among JA, SA, and ABA across biosynthetic and catabolic layers. Spatially, transcriptomic response overlap separated shoots from other tissues, with guard cells as shoot-side outliers showing weak JA–SA–ABA overlap. We further identified an SA-induced guard-cell-specific MYB60-centered module linked to ABA-associated stomatal regulators, suggesting a circuit that may fine-tune stomatal dynamics. Together, this atlas provides a high-resolution view of phytohormone response dynamics and interactions. Here the authors describe cell-type-specific responses of Arabidopsis seedlings to nine phytohormones. They characterize transcriptional crosstalk among them, and identify a guard-cell MYB60 module linked to stomatal regulation.
Zhijian Liu, Zhuowen Li, Yuzhuo Wang et al.· Nature Communications· 0 citations