Huanglongbing (HLB) is one of the most destructive diseases threatening global citrus production, yet how citrus plants respond to the pathogen at the cellular level has remained unclear. Using high-resolution single-nucleus transcriptomics, researchers uncovered how citrus roots reorganize their internal tissues when infected. The study reveals that infection suppresses phloem cell development—where the pathogen resides—while redirecting vascular differentiation toward lignified, defense-oriented xylem cells. This shift is accompanied by widespread activation of immune-related genes and metabolic pathways associated with cell wall strengthening. Together, these findings provide a cellular blueprint of how citrus plants balance survival and defense under severe disease pressure.
Huanglongbing (HLB), caused by the phloem-restricted bacterium Candidatus Liberibacter asiaticus, disrupts nutrient transport and ultimately leads to tree decline and yield loss. Previous studies have identified immune signaling pathways and pathogen effectors involved in disease progression, but most relied on bulk tissue analyses that mask differences between individual cell types. In reality, vascular tissues such as phloem, cambium, and xylem play distinct roles in disease susceptibility and resistance. Without cell-level resolution, it has been difficult to explain why certain citrus varieties tolerate infection better than others. Based on these challenges, there is a critical need to investigate citrus immune responses at single-cell resolution.
In a study published (DOI: 10.1093/hr/uhaf265) in 2025 in Horticulture Research, scientists from the Citrus Research Institute of Southwest University applied single-nucleus RNA sequencing to citrus roots infected with Candidatus Liberibacter asiaticus. By profiling thousands of individual cells, the team mapped how different vascular cell types respond during early stages of infection. Their analysis reveals profound changes in cell fate, gene expression, and tissue differentiation, offering the first single-cell atlas of citrus root immune responses to HLB.
The researchers generated single-nucleus transcriptomic profiles from rough lemon root tips, identifying 20 distinct cell clusters and five major vascular cell types. Across all cell types, infection triggered a coordinated immune response marked by the activation of transcription factors, pathogenesis-related proteins, and autophagy-associated genes. At the same time, genes involved in cytoskeleton organization and membrane signaling were broadly suppressed, suggesting a strategic reallocation of cellular resources.
A striking discovery emerged from developmental trajectory analysis. Infection significantly delayed the differentiation of cambium cells into phloem cells—the primary niche of the pathogen—while promoting their transition toward xylem cells. These xylem cells showed strong enrichment of stress-response genes and pathways involved in phenylpropanoid metabolism and lignin biosynthesis. Histological staining confirmed extensive lignin accumulation, indicating physical reinforcement of vascular tissues as a defense strategy.
The study also identified the transcription factor DOF2.4 as a potential regulator linking vascular development with immune activation. Manipulating its expression altered root structure, oxidative stress levels, and the expression of lignin- and flavonoid-related genes, highlighting its dual role in growth regulation and disease resistance.
“This work fundamentally changes how we understand citrus immunity,” said one of the study’s senior authors. “Rather than responding uniformly, citrus roots actively reprogram vascular development to limit pathogen spread. By suppressing vulnerable phloem tissues and reinforcing xylem cells, the plant creates a physical and biochemical barrier against infection. Identifying regulators such as DOF2.4 gives us new molecular entry points to enhance disease tolerance without compromising growth.”
These findings offer practical insights for citrus breeding and disease management. By pinpointing cell types and regulatory genes associated with tolerance, the study provides targets for molecular breeding, gene editing, and rootstock selection. Understanding how vascular tissues balance defense and development may also inform hormone-based or genetic strategies to mitigate long-term damage caused by HLB. Beyond citrus, the work demonstrates the power of single-cell technologies to reveal hidden defense mechanisms in perennial crops, paving the way for more resilient agricultural systems under increasing biotic stress.
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References
DOI
Original Source URL
https://doi.org/10.1093/hr/uhaf265
Funding information
This study was supported by the Rural Revitalization Project of Chinese Academy of Sciences (KCXFZJ-DDBF-202403), the Innovation Research 2035 Pilot Plan of Southwest University, China Agriculture Research System (CARS-26-05B), and the National Key R&D Program of China (Grant no. 2021YFD1400800).
About Horticulture Research
Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2023. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.
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