<b>DL</b><b>-</b><b>4</b><b>-</b><b>Chlorophenylalanine, a Novel Phytotoxin from</b><b> </b><b><i>Rhizoctonia</i></b><b><i> </i></b><b><i>solani</i></b><b> </b><b>AG3</b><b>-</b><b>TB,</b><b> </b><b>Induces Chloroplast</b><b> </b><b>Damage</b><b> </b><b>by</b><b> </b><b>Suppressing </b><b><i>NtDTX29</i></b><b>‑Mediated Detoxification in Tobacco</b>

<p dir="ltr">Tobacco target spot disease, caused by Rhizoctonia solani AG3-TB, results in leaf necrosis and chlorotic halo lesions primarily mediated by fungal phytotoxins. To identify the specific toxic components responsible for these pathological symptoms, an untargeted metabolomic profiling of the crude toxin revealed 510 chemical constituents, among which DL-4-chlorophenylalanine was confirmed through bioactivity assays as the principal pathogenic metabolite. Exposure of Nicotiana tabacum leaves to DL-4-chlorophenylalanine significantly increased reactive oxygen species (ROS) accumulation, reduced chlorophyll content in a dose-dependent manner, and disrupted chloroplast and mitochondrial membranes, consistent with toxin-induced necrosis. RNA-seq analysis of N. tabacum leaves treated for 48 h and 72 h identified 2,025 differentially expressed genes. Functional enrichment revealed perturbations in cell division, lipid metabolism, and antioxidant defense, while KEGG pathway analysis showed enrichment in MAPK signaling, peroxisome, plant-pathogen interaction, and hormone-signaling pathways, indicating activation of multifaceted defense networks against toxin stress. Among the down-regulated genes, DETOXIFICATION 29 (NtDTX29), a member of the MATE transporter family, was identified as a critical component conferring tolerance. Overexpression of NtDTX29 in N. benthamiana markedly enhanced tolerance to both the crude toxin and purified DL-4-chlorophenylalanine, suggesting a role in active toxin efflux and preservation of organelle integrity. These findings provide new insight into the chemical and molecular basis of R. solani AG3-TB pathogenicity and tobacco defense mechanisms. The elucidated detoxification pathway offers theoretical guidance for breeding or engineering toxin-resistant tobacco varieties and for developing targeted, environmentally friendly strategies to manage tobacco target spot disease sustainably.</p>