Abstract
Perception of pathogen-associated molecular patterns (PAMPs), such as bacterial flagellin (or the peptide flg22), by surface-localized receptors activates defense responses and subsequent immunity. In a previous forward-genetic screen aimed at the identification of Arabidopsis (Arabidopsis thaliana) flagellin-insensitive (fin) mutants, we isolated fin4, which is severely affected in flg22-triggered reactive oxygen species (ROS) bursts. Here, we report that FIN4 encodes the chloroplastic enzyme ASPARTATE OXIDASE (AO), which catalyzes the first irreversible step in the de novo biosynthesis of NAD. Genetic studies on the role of NAD have been hindered so far by the lethality of null mutants in NAD biosynthetic enzymes. Using newly identified knockdown fin alleles, we found that AO is required for the ROS burst mediated by the NADPH oxidase RBOHD triggered by the perception of several unrelated PAMPs. AO is also required for RBOHD-dependent stomatal closure. However, full AO activity is not required for flg22-induced responses that are RBOHD independent. Interestingly, although the fin4 mutation dramatically affects RBOHD function, it does not affect functions carried out by other members of the RBOH family, such as RBOHC and RBOHF. Finally, we determined that AO is required for stomatal immunity against the bacterium Pseudomonas syringae. Altogether, our work reveals a novel specific requirement for AO activity in PAMP-triggered RBOHD-dependent ROS burst and stomatal immunity. In addition, the availability of viable mutants for the chloroplastic enzyme AO will enable future detailed studies on the role of NAD metabolism in different cellular processes, including immunity, in Arabidopsis.
Highlights
Perception of pathogen-associated molecular patterns (PAMPs), such as bacterial flagellin, by surfacelocalized receptors activates defense responses and subsequent immunity
We report that FIN4 encodes the chloroplastic enzyme ASPARTATE OXIDASE (AO), which catalyzes the first irreversible step in the de novo biosynthesis of nicotinamide adenine dinucleotide (NAD). fin4 corresponds to a viable knockdown mutant that is impaired in the RBOHD-dependent PAMP-induced reactive oxygen species (ROS) burst and stomatal closure
NAD is an essential cofactor in energy metabolism and electron transfer
Summary
Research School of Biology, Australian National University, Canberra, Australian Capital Territory 0200, Australia. RBOHD is the main enzyme responsible for the rapid production of apoplastic ROS upon PAMP perception (Nühse et al, 2007; Zhang et al, 2007). Apoplastic peroxidases, such as PRX33 and PRX34, have been shown to contribute to flg22- and elf26-triggered ROS bursts (Daudi et al, 2012; O’Brien et al, 2012). Supporting the importance of stomatal closure for plant immunity, a COR-deficient (COR2) derivative of the Arabidopsis pathogen P. syringae pv tomato (Pto) DC3000 is unable to colonize the apoplast efficiently or cause disease when surface inoculated (Melotto et al, 2006). Fin corresponds to a viable knockdown mutant that is impaired in the RBOHD-dependent PAMP-induced ROS burst and stomatal closure.
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