AUTHOR=Imano Sayaka , Fushimi Mayuka , Camagna Maurizio , Tsuyama-Koike Akiko , Mori Hitoshi , Ashida Akira , Tanaka Aiko , Sato Ikuo , Chiba Sotaro , Kawakita Kazuhito , Ojika Makoto , Takemoto Daigo 

TITLE=AP2/ERF Transcription Factor NbERF-IX-33 Is Involved in the Regulation of Phytoalexin Production for the Resistance of Nicotiana benthamiana to Phytophthora infestans

JOURNAL=Frontiers in Plant Science

VOLUME=12

YEAR=2022

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2021.821574

DOI=10.3389/fpls.2021.821574

ISSN=1664-462X

ABSTRACT=<p>Plants recognize molecular patterns unique to a certain group of microbes to induce effective resistance mechanisms. Elicitins are secretory proteins produced by plant pathogenic oomycete genera including <italic>Phytophthora</italic> and <italic>Pythium.</italic> Treatment of INF1 (an elicitin produced by <italic>P. infestans</italic>) induces a series of defense responses in <italic>Nicotiana</italic> species, including reactive oxygen species (ROS) production, transient induction of ethylene production, hypersensitive cell death and accumulation of the sesquiterpenoid phytoalexin capsidiol. In this study, we analyzed the expression profiles of <italic>N. benthamiana</italic> genes after INF1 treatment by RNAseq analysis. Based on their expression patterns, <italic>N. benthamiana</italic> genes were categorized into 20 clusters and 4,761 (8.3%) out of 57,140 genes were assigned to the clusters for INF1-induced genes. All genes encoding enzymes dedicated to capsidiol production, 5-<italic>epi</italic>-aristolochene (EA) synthase (<italic>NbEAS</italic>, 10 copies) and EA dehydrogenase (<italic>NbEAH</italic>, 6 copies), and some genes for ethylene production, such as 1-aminocyclopropane 1-carboxylate (ACC) synthase (<italic>NbACS</italic>) and ACC oxidase (<italic>NbACO</italic>), were significantly upregulated by INF1 treatment. Analysis of <italic>NbEAS1</italic> and <italic>NbEAS4</italic> promoters revealed that AGACGCC (GCC box-like motif) is the essential cis-element required for INF1-induced expression of <italic>NbEAS</italic> genes. Given that the GCC box is known to be targeted by ERF (ethylene-responsive factor) transcription factors, we created a complete list of <italic>N. benthamiana</italic> genes encoding AP2/ERF family transcription factors, and identified 45 out of 337 <italic>AP2/ERF</italic> genes in the clusters for INF1-induced genes. Among INF1-induced <italic>NbERF</italic> genes, silencing of <italic>NbERF-IX-33</italic> compromised resistance against <italic>P. infestans</italic> and INF1-induced production of capsidiol. Recombinant NbERF-IX-33 protein can bind to the promoter sequence of <italic>NbEAS4</italic>, suggesting that NbERF-IX-33 is a transcription factor directly regulating the expression of genes for phytoalexin production.</p>