Abstract

   During the early systemic infection of plant pathogens, individual
   cells can harbor pathogens at various stages of infection, ranging from
   absent to abundant. Consequently, gene expression levels within these
   cells in response to the pathogens exhibit significant variability.
   These variations are pivotal in determining pathogenicity or
   susceptibility, yet they remain largely unexplored and poorly
   understood. Sugarcane mosaic virus (SCMV) is a representative member of
   the monocot-infecting potyviruses with a polyadenylated RNA genome,
   which can be captured by single-cell RNA sequencing (scRNA-seq). Here,
   we performed scRNA-seq on SCMV-infected maize leaves during early
   systemic infection (prior to symptom manifestation) to investigate the
   co-variation patterns between viral accumulation and intracellular gene
   expression alterations. We identified five cell types and found that
   mesophyll-4 (MS4) cells exhibited the highest levels of viral
   accumulation in most cells. Early systemic infection of SCMV resulted
   in a greater upregulation of differentially expressed genes, which were
   mainly enriched in biological processes related to translation, peptide
   biosynthesis, and metabolism. Co-variation analysis of the altered
   maize gene expression and viral accumulation levels in MS1, 2, and 4
   revealed several patterns, and the co-expression relationships between
   them were mainly positive. Furthermore, functional studies identified
   several potential anti- or pro-viral factors that may play crucial
   roles during the early stage of SCMV systemic infection. These results
   not only provide new insights into plant gene regulation during viral
   infection but also offer a foundation for future investigations of
   host–virus interactions across molecular, cellular, and physiological
   scales.

   Key words: systemic infection, differentially expressed genes,
   co-variation, functional study, anti- or pro-viral factors
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   Our understanding of plant-virus pathogenic mechanisms and host gene
   regulation during the initial phase of systemic infection remains
   limited. This study investigates gene expression patterns in maize at
   the single-cell level during early systemic infection of SCMV,
   revealing patterns of co-variation between viral loads and alterations
   in maize gene expression, as well as identifying anti- and pro-viral
   factors.

Introduction

   Plant viral systemic infections cause severe damage to crops, which can
   lead to epidemics and pose serious threats to food security. In plants,
   viral infection is a dynamic process driven by the interplay between
   antiviral cellular pathways and viral machinery ([45]Calil and Fontes,
   2017; [46]Fontes et al., 2021). Following initial infection, this
   process progresses through multiple stages. The virus first invades the
   plant cell and releases its own genetic material (RNA or DNA) for
   replication, with increasing levels of viral accumulation depending on
   the order of viral invasion. Simultaneously, the virus hijacks the
   host’s protein synthesis system to synthesizes viral proteins in large
   quantities. Subsequently, the virus moves from initial infection sites
   in epidermal or phloem cells (for insect-borne viruses) into the sieve
   elements for long-distance movement. The virus then exits the sieve
   elements in systemically infected tissues by crossing the bundle sheath
   (BS), vascular parenchyma, and companion cells ([47]Seo and Kim, 2016;
   [48]Xue et al., 2023). As the virus moves systemically, it can
   replicate and/or assemble in many cells to efficiently establish
   systemic infection. Since virus-induced changes in plant gene
   expression during the early stages of systemic infection (before
   symptoms manifest) are key drivers of pathogenesis, understanding these
   alterations and their roles in viral infection is critical for
   developing novel virus control strategies.

   Maize (Zea mays L.), one of the most productive and widely cultivated
   crops globally, plays an increasingly important role in food production
   ([49]Pechanova and Pechan, 2015). Maize production is threatened by
   both abiotic and biotic stresses, including viral infections. Sugarcane
   mosaic virus (SCMV) is a prevalent monocot-infecting potyvirus that
   causes maize dwarf mosaic disease (MDMD) in many maize-producing
   regions of China, Europe, and Africa ([50]Jiang and Zhou, 2002; [51]Fan
   et al., 2003; [52]Mahuku et al., 2015; [53]Redinbaugh and Stewart,
   2018; [54]Braidwood et al., 2019). Over the past several decades,
   intensive efforts have explored potential measures for controlling SCMV
   and MDMD ([55]Kannan et al., 2018). However, the molecular events that
   are crucial to the infection process and pathogenicity of SCMV in maize
   remain largely elusive.

   Recently, we characterized the manifestation of mosaic symptoms
   following systemic SCMV infection in maize ([56]Jiang et al., 2023).
   During its distal movement, SCMV travels upward through the vascular
   system, with mosaic symptoms first appearing at the base of upper
   leaves ([57]Du et al., 2020; [58]Jiang et al., 2023). The virus then
   continues to spread, and symptoms emerge in most regions of
   systemically infected leaves. Consequently, during early systemic
   infection—before symptoms are visible—the virus enters new cells and
   begins to multiply, with viral accumulation levels varying widely from
   absent to high concentrations within different cells of the same leaf.
   This dynamic process of early systemic SCMV infection in maize provides
   a suitable study system to explore the patterns of co-variation between
   viral accumulation and changes in host gene expression at the
   single-cell level.

   Previous omics studies analyzing tissues, organs, or entire organisms,
   treated samples as homogeneous bulk material, averaging out any
   variation present among cells within them. The recent development of
   single-cell RNA sequencing (scRNA-seq) technology makes it possible to
   resolve cell-to-cell transcriptomic heterogeneity and has been applied
   to numerous plant species (e.g., Arabidopsis, maize, rice, tomato, and
   cotton; reviewed in [59]Cuperus, 2022; [60]Ryu et al., 2021;
   [61]Seyfferth et al., 2021), providing new perspectives on gene
   expression and cell-type evolution in plants. Single-cell
   characterization of gene expression has been performed on multiple
   tissues in maize, including leaves ([62]Bezrutczyk et al., 2021;
   [63]Sun et al., 2022; [64]Tao et al., 2022), shoot apical meristems
   ([65]Satterlee et al., 2020), roots ([66]Ortiz-Ramirez et al., 2021;
   [67]Li et al., 2022; [68]Cao et al., 2023; [69]Guillotin et al., 2023),
   anthers ([70]Nelms and Walbot, 2019; [71]Zhang et al., 2021;
   [72]Washburn et al., 2023), ears ([73]Xu et al., 2021), and seedlings
   ([74]Marand et al., 2021), providing valuable references for this