Mechanisms underneath Xanthomonas citri subsp. citri promoting of Agrobacterium-mediated transient expression in citrus

Agrobacterium-mediated transient expression (TE) is an important tool in plant genetics study and biotechnology. Citrus is one of the top tree crops, but Agrobacterium-mediated TE remains problematic in citrus. Previous study has shown that pretreatment of citrus leaves with Xanthomonas citri subsp. citri (Xcc), which causes citrus canker in almost all citrus cultivars, significantly improves the TE efficacy. Here we have shown that Xcc promotes Agrobacterium- mediated TE via improving Agrobacterium growth, suppressing plant immune response, triggering cell division, and upregulating plant cell wall degrading enzymes. We demonstrate that Xcc promotes Agrobacterium-mediated TE via PthA and mutation of pthA4 abolishes the promoting effect of Xcc. PthA4 is known to trigger cell division of citrus. We have demonstrated the causative relationship between cell division and Agrobacterium-mediated TE using cell division inhibitor mimosine. Importantly, the differential effects of mimosine and colchicine on Xcc promoting Agrobacterium-mediated TE indicate that S-phase is critical for agroinfiltration of citrus tissues. In addition, PthA4 is known to upregulate plant growth hormones auxin (IAA), gibberellin and cytokinin, as well as cell degrading enzymes (e.g., cellulase). Exogenous application of IAA, cytokinin, and cellulase but not gibberellin significantly improved Agrobacterium-mediated TE in leaves of sweet orange, pummelo, Meiwa kumquat, lucky bamboo and rose mallow. Our study provides a mechanistic understanding regarding how Xcc promotes Agrobacterium-mediated TE and identified two practical measures to improve Agrobacterium-mediated TE via pretreatment with plant hormones (i.e., auxin and cytokinin) and cellulase.