A peroxidase-dependent apoplastic oxidative burst in cultured Arabidopsis cells functions in MAMP-elicited defense.
Perception by plants of so-called microbe-associated molecular patterns (MAMPs) such as bacterial flagellin, referred to as pattern-triggered immunity, triggers a rapid transient accumulation of reactive oxygen species (ROS). We previously identified two cell wall peroxidases, PRX33 and PRX34, involved in apoplastic hydrogen peroxide (H2O2) production in Arabidopsis (Arabidopsis thaliana). Here, we describe the generation of Arabidopsis tissue culture lines in which the expression of PRX33 and PRX34 is knocked down by antisense expression of a heterologous French bean (Phaseolus vulgaris) peroxidase cDNA construct. Using these tissue culture lines and two inhibitors of ROS generation, azide and diphenylene iodonium, we found that perxoxidases generate about half of the H2O2 that accumulated in response to MAMP treatment and that NADPH oxidases and other sources such as mitochondria account for the remainder of the ROS. Knockdown of PRX33/PRX34 resulted in decreased expression of several MAMP-elicited genes, including MYB51, CYP79B2, and CYP81F2. Similarly, proteomic analysis showed that knockdown of PRX33/PRX34 led to the depletion of various MAMP-elicited defense-related proteins, including the two cysteine-rich peptides PDF2.2 and PDF2.3. Knockdown of PRX33/PRX34 also led to changes in the cell wall proteome, including increases in enzymes involved in cell wall remodeling, which may reflect enhanced cell wall expansion as a consequence of reduced H2O2-mediated cell wall cross-linking. Comparative metabolite profiling of a CaCl2 extract of the PRX33/PRX34 knockdown lines showed significant changes in amino acids, aldehydes, and keto acids but not fatty acids and sugars. Overall, these data suggest that PRX33/PRX34-generated ROS production is involved in the orchestration of pattern-triggered immunity in tissue culture cells.
School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, United Kingdom.
Jose A
JA
O'Brien
Arsalan
A
Daudi
Paul
P
Finch
Vernon S
VS
Butt
Julian P
JP
Whitelegge
Puneet
P
Souda
Frederick M
FM
Ausubel
G Paul
GP
Bolwell
D017360:Arabidopsis / Q000166:cytology* / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002473:Cell Wall / Q000187:drug effects / Q000201:enzymology; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D042541:Intracellular Space / Q000187:drug effects / Q000201:enzymology*; D019255:NADPH Oxidases / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology; D010544:Peroxidases / Q000235:genetics / Q000378:metabolism*; D027805:Phaseolus / Q000187:drug effects / Q000201:enzymology; D057865:Plant Immunity / Q000187:drug effects; D030821:Plants, Genetically Modified; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D051192:Receptors, Pattern Recognition / Q000276:immunology*; D016897:Respiratory Burst / Q000187:drug effects; D019810:Sodium Azide / Q000633:toxicity
D029681:Arabidopsis Proteins; D018076:DNA, Complementary; D009861:Onium Compounds; D012333:RNA, Messenger; D051192:Receptors, Pattern Recognition; C007517:diphenyleneiodonium; D019810:Sodium Azide; D006861:Hydrogen Peroxide; D010544:Peroxidases; C574008:PRX33 protein, Arabidopsis; C553803:PRX34 protein, Arabidopsis; C568857:AT5G47910 protein, Arabidopsis; D019255:NADPH Oxidases