Unlocking Phytoalexin Biosynthesis Using Multigene Engineering

Abstract

Plants have evolved sophisticated defense mechanisms to balance growth and immunity, with phytoalexins and lignin playing central roles in pathogen resistance. In Arabidopsis thaliana, the jasmonate repressor JAZ1 and transcription factor ANAC042 act as opposing regulators of this process. The regulation of phytoalexin biosynthesis is highly complex, and manipulating individual transcription factors has generally proven insufficient to fully activate the pathway. To address this limitation, this study investigates whether the knockout of JAZ1 in combination with the overexpression of ANAC042 can more effectively unlock phytoalexin biosynthesis. While JAZ1 knockout promoted systemic lignification under prolonged stress, while ANAC042 overexpression alone did not strongly induce phytoalexins. However, their combination enhanced accumulation of hydroxyindole-3-carbonyl nitrile and monolignols. Transcriptional analysis revealed altered regulation of WRKY33 and MYB15, suggesting that JAZ1 and ANAC042 are a part of a broader defense regulatory network. Growth-defense trade-offs showed growth penalties in all tested genotypes under Flg22 elicitation, except in ANAC042 overexpression lines, where growth was unaffected by the elicitor, highlighting the metabolic costs associated with sustained immunity. Extending these findings to soybean, a dual-gene plasmid (pGEMINI-B) was constructed to co-overexpress two positive regulators of glyceollin biosynthesis, while silencing GmJAZ1 genes. Collectively, these findings demonstrate that manipulating antagonistic regulatory factors simultaneously can unlock levels of phytoalexin accumulation that are unattainable through single-gene modifications. The underlying shifts in gene expression and metabolism driving this effect are unconventional and warrant deeper investigation, as elevated phytoalexin biosynthetic gene expression does not directly account for the observed increases in phytoalexin levels.