Exploration of the Interactions Between Xanthomonas citri subsp. citri-Agrobacterium-Citrus to Improve Agrobacterium-Mediated Transient Expression in Plants.

Agrobacterium-mediated transient expression (AMTE) is an important tool in plant genetics studies and biotechnology. AMTE remains problematic in citrus and many plant species. Previous research has shown that pretreatment of citrus leaves with Xanthomonas citri subsp. citri (Xcc), which causes citrus canker, significantly improves the AMTE efficacy. Here, we have shown that Xcc promotes AMTE mainly through triggering cell division and upregulating plant cell wall-degrading enzymes. We demonstrate that Xcc improves AMTE via PthA, a transcription activator-like effector known to trigger cell division in citrus, and mutation of pthA4 abolished the promoting effect of Xcc. Mutation of the effector (PthA4)-binding element in the promoter region and coding region of CsLOB1, which is known to be directly activated by PthA4, significantly reduced Xcc promotion of AMTE. Mutation of PthA4 significantly reduced the expression of cell division-related genes (CDKA, CDKB1-2, and CDKB2-2) compared with wild-type Xcc and the complemented strain. Cell division inhibitor mimosine but not colchicine also significantly decreased Xcc promotion of AMTE. In addition, PthA4 is known to upregulate plant growth hormones auxin (indole-3-acetic acid), gibberellin, and cytokinin, as well as cell wall-degrading enzymes (e.g., cellulase). Exogenous application of indole-3-acetic acid, cytokinin, and cellulase but not gibberellin significantly improved AMTE in leaves of sweet orange, pummelo, Meiwa kumquat, lucky bamboo, and rose mallow. Our study provides a mechanistic understanding of how Xcc promotes AMTE and develops practical measures to improve AMTE via pretreatment with plant hormones (i.e., auxin and cytokinin) and cellulase. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.