AUTHOR=Liu Tiantian , Zhou Zehua , Luo Changwei , Luo Hua , Tang Jun , Shi Xiaojiang , Li Diping , Zhang Qiong , Li Jin , Xia Yonggang , Song Na , Yi Tuyong 

TITLE=Elucidation of mechanisms underlying active oxygen burst in Citrus sinensis after Diaporthe citri infection using transcriptome analysis

JOURNAL=Frontiers in Microbiology

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2024.1425441

DOI=10.3389/fmicb.2024.1425441

ISSN=1664-302X

ABSTRACT=<sec id="sec1"><title>Introduction</title><p>Reactive oxygen species (ROS) generation is a common disease defense mechanism in plants. However, it is unclear whether <italic>Citrus</italic> host activates defense response against <italic>Diaporthe citri</italic> causing citrus melanose disease by producing ROS, and the underlying molecular mechanisms are unknown.</p></sec><sec id="sec2"><title>Methods</title><p>DAB staining and RNA-Seq technology were used to compare the active oxygen burst and differential gene expression, respectively, in uninfected and infected <italic>Citrus sinensis</italic> leaves at different time points during <italic>D. citri</italic> infection <italic>in vivo</italic>. The functions of <italic>CsRBOH</italic> (a significant DEG) were confirmed in <italic>N. benthamiana</italic> through the <italic>Agrobacterium</italic>-mediated transient expression system.</p></sec><sec id="sec3"><title>Results</title><p>DAB staining indicated that <italic>C. sinensis</italic> initiated defense against <italic>D. citri</italic> infection within 24 h by generating ROS. Illumina sequencing revealed 25,557 expressed genes of <italic>C. sinensis</italic>. The most upregulated DEGs (<italic>n</italic> = 1,570) were identified 72 h after fungal inoculation (sample denoted as CD72). In the CD72 vs. Cs (samples at 0 h after fungal inoculation) comparison, the KEGG pathway category with the highest number of genes (<italic>n</italic> = 62) and most significant enrichment was Protein processing in endoplasmic reticulum, followed by Glutathione metabolism and MAPK signaling pathway-plant. GO analysis revealed that the DEGs of CD72 vs. Cs related to active oxygen burst and chitin recognition were significantly grouped into the regulation of biological processes and molecular functions, with GO terms including response to ROS, response to fungus, and oxidoreductase activity. Remarkably, <italic>CsRBOH</italic> was significantly enriched in the GO and KEGG analyses, and its expression pattern in qRT-PCR and DAB staining results were consistent. Among the 63 ROS-related DEGs, HSP genes and genes associated with the peroxidase family were highly significant as revealed by protein–protein interaction networks. Furthermore, ROS accumulation, cell death, and upregulation of defense-related genes were observed in <italic>N. benthamiana</italic> leaves with <italic>CsRBOH</italic> expressed through the <italic>Agrobacterium</italic>-mediated transient expression system.</p></sec><sec id="sec4"><title>Conclusion</title><p>Our findings suggested that <italic>C. sinensis</italic> activates <italic>CsRBOH</italic> and ROS-related genes, leading to ROS accumulation to resist the invasion by <italic>D. citri</italic>. This study laid the foundation for future research on molecular mechanisms and breeding of <italic>C. sinensis</italic> cultivars resistant to citrus melanose.</p></sec>