TY - JOUR
T1 - Lumi-Map, a real-time luciferase bioluminescence screen of mutants combined with MutMap, reveals Arabidopsis genes involved in PAMP-triggered immunity
AU - Kato, Hiroaki
AU - Onai, Kiyoshi
AU - Abe, Akira
AU - Shimizu, Motoki
AU - Takagi, Hiroki
AU - Tateda, Chika
AU - Utsushi, Hiroe
AU - Singkarabanit-Ogawa, Suthitar
AU - Kitakura, Saeko
AU - Ono, Erika
AU - Zipfel, Cyril
AU - Takano, Yoshitaka
AU - Ishiura, Masahiro
AU - Terauchi, Ryoehei
PY - 2020/12
Y1 - 2020/12
N2 - Plants recognize pathogen-associated molecular patterns (PAMPs) to activate PAMP-triggered immunity (PTI). However, our knowledge of PTI signaling remains limited. In this report, we introduce Lumi-Map, a high-throughput platform for identifying causative single-nucleotide polymorphisms (SNPs) for studying PTI signaling components. In Lumi-Map, a transgenic reporter plant line is produced that contains a firefly luciferase (LUC) gene driven by a defense gene promoter, which generates luminescence upon PAMP treatment. The line is mutagenized and the mutants with altered luminescence patterns are screened by a high-throughput real-time bioluminescence monitoring system. Selected mutants are subjected to MutMap analysis, a whole-genome sequencingbased method of rapid mutation identification, to identify the causative SNP responsible for the luminescence pattern change. We generated nine transgenic Arabidopsis reporter lines expressing the LUC gene fused to multiple promoter sequences of defense-related genes. These lines generate luminescence upon activation of FLAGELLIN-SENSING 2 (FLS2) by flg22, a PAMP derived from bacterial flagellin. We selected the WRKY29-promoter reporter line to identify mutants in the signaling pathway downstream of FLS2. After screening 24,000 ethylmethanesulfonate-induced mutants of the reporter line, we isolated 22 mutants with altered WRKY29 expression upon flg22 treatment (abbreviated as awf mutants). Although five flg22-insensitive awf mutants harbored mutations in FLS2 itself, Lumi-Map revealed three genes not previously associated with PTI. Lumi-Map has the potential to identify novel PAMPs and their receptors as well as signaling components downstream of the receptors.
AB - Plants recognize pathogen-associated molecular patterns (PAMPs) to activate PAMP-triggered immunity (PTI). However, our knowledge of PTI signaling remains limited. In this report, we introduce Lumi-Map, a high-throughput platform for identifying causative single-nucleotide polymorphisms (SNPs) for studying PTI signaling components. In Lumi-Map, a transgenic reporter plant line is produced that contains a firefly luciferase (LUC) gene driven by a defense gene promoter, which generates luminescence upon PAMP treatment. The line is mutagenized and the mutants with altered luminescence patterns are screened by a high-throughput real-time bioluminescence monitoring system. Selected mutants are subjected to MutMap analysis, a whole-genome sequencingbased method of rapid mutation identification, to identify the causative SNP responsible for the luminescence pattern change. We generated nine transgenic Arabidopsis reporter lines expressing the LUC gene fused to multiple promoter sequences of defense-related genes. These lines generate luminescence upon activation of FLAGELLIN-SENSING 2 (FLS2) by flg22, a PAMP derived from bacterial flagellin. We selected the WRKY29-promoter reporter line to identify mutants in the signaling pathway downstream of FLS2. After screening 24,000 ethylmethanesulfonate-induced mutants of the reporter line, we isolated 22 mutants with altered WRKY29 expression upon flg22 treatment (abbreviated as awf mutants). Although five flg22-insensitive awf mutants harbored mutations in FLS2 itself, Lumi-Map revealed three genes not previously associated with PTI. Lumi-Map has the potential to identify novel PAMPs and their receptors as well as signaling components downstream of the receptors.
KW - Arabidopsis thaliana
KW - Elicitors
KW - Genetics and gene regulation
KW - High-throughput phenotyping
KW - Luciferase reporter
KW - MAMPs
KW - MutMap
KW - PAMP-triggered immunity
KW - Plant defense mechanisms
KW - Receptor
UR - http://www.scopus.com/inward/record.url?scp=85097211507&partnerID=8YFLogxK
U2 - 10.1094/MPMI-05-20-0118-TA
DO - 10.1094/MPMI-05-20-0118-TA
M3 - Article
VL - 33
SP - 1366
EP - 1380
JO - Molecular Plant-Microbe Interactions
JF - Molecular Plant-Microbe Interactions
SN - 0894-0282
IS - 12
ER -