The effector AvrRxo1 phosphorylates NAD in planta.

Area of Science:

• Microbiology
• Plant Pathology
• Biochemistry

Background:

• Gram-negative bacteria use type III secreted effectors to suppress host immunity.
• AvrRxo1, a Xanthomonas effector, is a homolog of sugar-nucleotide kinases and suppresses bacterial growth.
• Previous studies suggested AvrRxo1 might manipulate primary metabolic pathways by phosphorylating NAD.

Purpose of the Study:

• To investigate the in planta activity of AvrRxo1.
• To determine if AvrRxo1 phosphorylates NAD in plants and if this activity is essential for its functions.
• To identify the product of AvrRxo1-mediated phosphorylation and its role in virulence and immunity suppression.

Main Methods:

• Global metabolomics to identify phosphorylation products.
• In vitro kinase assays to confirm NAD kinase activity.
• Site-directed mutagenesis to assess the role of catalytic sites and ATP-binding motifs.

Main Results:

• AvrRxo1 was confirmed to phosphorylate NAD in planta, producing 3 -NADP.
• 3 -NADP accumulated in yeast, bacteria, and plant tissues expressing AvrRxo1.
• Mutation of the catalytic residue D193 abolished kinase activity and key AvrRxo1 phenotypes.
• Mutation in the Walker A motif affected toxicity but not NAD phosphorylation or virulence enhancement.

Conclusions:

• AvrRxo1 functions as an NAD kinase in planta, targeting the central metabolite NAD.
• The kinase activity of AvrRxo1 is essential for its toxicity and suppression of the reactive oxygen species (ROS) burst.
• AvrRxo1 manipulates host metabolism and immunity through NAD phosphorylation, highlighting a novel virulence strategy.