The Halophyte Halostachys caspica miR172e/TOE3 module regulates Arabidopsis resistance to Pst DC3000 and Verticillium dahlia

Upon pathogen invasion, plants activate sophisticated immune signaling cascades to defense against pathogenic attack. AP2/ERF transcription factors function as crucial regulators of plant immune responses. Halostachys caspica , an exceptionally salt-tolerant halophyte belonging to Amaranthaceae, is widely distributed in the arid and saline-alkaline regions of Xinjiang, Northwest China. In this study, we investigated the effects of HcmiR172e and its target gene HcTOE3 , an the AP2 subfamily transcription factor from H. caspica , in regulating plant responses to Pseudomonas syringae pv. tomato DC3000 ( Pst DC3000) and Verticillium dahliae (VD991) infection. The results revealed that HcTOE3 was transcriptionally induced by Pst DC3000, VD991, and multiple phytohormonal treatments. Compared with wild-type (Col-0) plants, HcTOE3- overexpressing transgenic Arabidopsis plants exhibited enhanced resistance to Pst DC3000 and VD991 infections, accompanied by increased reactive oxygen species (ROS) accumulation, improved photosynthetic performance, and reduced pathogen proliferation. Notably, following Pst DC3000 infection, HcTOE3 triggers rapid and transient stomatal immune response and activates the expression of AtPAD4 that plays an important role in involving phytoalexin biosynthesis. In response to VD991 infection, HcTOE3 significantly enhanced lignin accumulation in roots by upregulating key genes involved in the phenylpropanoid pathway, including AtPAL4 and AtC4H . Furthermore, HcTOE3 significantly activated the transcription of multiple defense-related genes associated with salicylic acid (SA)- and jasmonic acid (JA)-mediated signaling pathways, including AtPR1 , AtPR2 , AtPR5 , AtPDF1.2 , and AtVSP2 , as well as defense marker genes such as AtWRKY53 , AtERF104 , and AtPAD4 . In contrast, HcmiR172-eoverespressing Arabidopsis plants exhibited opposite molecular and physiological phenotypes, indicating that HcmiR172e negatively regulates plant immunity by suppressing HcTOE3 expression. Yeast one-hybrid and dual-luciferase reporter analyses demonstrated that HcTOE3 directly binds to the promoter regions of downstream defense-associated genes, including AtPR1 , AtPDF1.2 , AtPR2 , and AtVSP2 . This transcriptional activation depends on the recognition of spectific cis-acting elements, including TTTGTT, AGCTA, GAAG, and MYB-binding motifs, thereby facilitating the activation of plant defense responses. Taken together, these findings demonstrate that HcTOE3 positively regulates Arabidopsis resistance to Pst DC3000 and VD991, whereas HcmiR172e functions as a negative regulator of plant immune defense. This study provides novel mechanistic insights and empirical evidence for understanding the regulatory functions of AP2 subfamily transcription factors and highlights the potential of halophyte-derived immune regulators for improving crop resistance against biotic stresses.