Mutant Allele-Specific Uncoupling of PENETRATION3 Functions Reveals Engagement of the ATP-Binding Cassette Transporter in Distinct Tryptophan Metabolic Pathways

Abstract

Arabidopsis (Arabidopsis thaliana) PENETRATION (PEN) genes quantitatively contribute to the execution of different forms of plant immunity upon challenge with diverse leaf pathogens. PEN3 encodes a plasma membrane-resident pleiotropic drug resistance-type ATP-binding cassette transporter and is thought to act in a pathogen-inducible and PEN2 myrosinasedependent metabolic pathway in extracellular defense. This metabolic pathway directs the intracellular biosynthesis and activation of tryptophan-derived indole glucosinolates for subsequent PEN3-mediated efflux across the plasma membrane at pathogen contact sites. However, PEN3 also functions in abiotic stress responses to cadmium and indole-3-butyric acid (IBA)-mediated auxin homeostasis in roots, raising the possibility that PEN3 exports multiple functionally unrelated substrates. Here, we describe the isolation of a pen3 allele, designated pen3-5, that encodes a dysfunctional protein that accumulates in planta like wild-type PEN3. The specific mutation in pen3-5 uncouples PEN3 functions in IBA-stimulated root growth modulation, callose deposition induced with a conserved peptide epitope of bacterial flagellin (flg22), and pathogen-inducible salicylic acid accumulation from PEN3 activity in extracellular defense, indicating the engagement of multiple PEN3 substrates in different PEN3-dependent biological processes. We identified 4-O-b-D-glucosyl-indol-3-yl formamide (4OGlcI3F) as a pathogen-inducible, tryptophan-derived compound that overaccumulates in pen3 leaf tissue and has biosynthesis that is dependent on an intact PEN2 metabolic pathway. We propose that a precursor of 4OGlcI3F is the PEN3 substrate in extracellular pathogen defense. These precursors, the shared indole core present in IBA and 4OGlcI3F, and allele-specific uncoupling of a subset of PEN3 functions suggest that PEN3 transports distinct indole-type metabolites in distinct biological processes.

Arabidopsis ABCG34 contributes to defense against necrotrophic pathogens by mediating the secretion of camalexin

“…Two different classes of such chemicals may be involved in this process: surface-coating materials, as shown for AtABCG32/PEC1 (21,22), and secondary metabolites, as shown for AtABCG36/PDR8/ PEN3 (14). We tested the first possibility by an ethanol penetration assay that detects permeability defects but could not observe any difference in ethanol penetration between the wildtype plants and atabcg34 mutants (Fig.…”

“…PDR8/ ABCG36/PEN3, which is localized at the plasma membrane, recruited to infection sites (11), and involved in glucosinolatedependent pathogen defense at the contact site (12), blocks the penetration of nonhost fungal pathogens (13). Recently, a report suggested that this protein transports 4-O-β-D-glucosyl-indol-3-yl formamide (4OGlcI3F) (14); however, this possibility remains to be proven. atabcg40/pdr12 mutants exhibit sensitivity to sclareol, and AtABCG40/PDR12 is induced by pathogen inoculation (15).…”

Immobilized Subpopulations of Leaf Epidermal Mitochondria Mediate PENETRATION2-Dependent Pathogen Entry Control in Arabidopsis

“…Together, these data support a scenario in which PEN3 mediates the ATP-dependent transport of glucosinolates that were enzymatically activated by PEN2 and are potentially toxic for invading pathogens (Stein et al, 2006). PEN2 was shown to hydrolyze indol-3-ylmethylglucosinolate (I3G) and 4-methoxyindol-3-ylmethylglucosinolate (4MI3G) (Bednarek et al, 2009) and to be required for production of 4-O-b-D-glucosylindol-3-yl formamide (Lu et al, 2015). Deglucosylated precursor molecules of the latter have recently been proposed to represent the critical PEN3 transport substrate in powdery mildew entry control (Lu et al, 2015).…”

“…PEN2 was shown to hydrolyze indol-3-ylmethylglucosinolate (I3G) and 4-methoxyindol-3-ylmethylglucosinolate (4MI3G) (Bednarek et al, 2009) and to be required for production of 4-O-b-D-glucosylindol-3-yl formamide (Lu et al, 2015). Deglucosylated precursor molecules of the latter have recently been proposed to represent the critical PEN3 transport substrate in powdery mildew entry control (Lu et al, 2015). As I3G and 4MI3G contain S-glycosidic bonds, PEN2 represents a thioglucosidase (myrosinase) but was designated as an atypical myrosinase due to an unusual amino acid composition within the catalytic center.…”

Pathogen-Responsive MPK3 and MPK6 Reprogram the Biosynthesis of Indole Glucosinolates and Their Derivatives in Arabidopsis Immunity

“…Recently, 4-O-b-D-glucosyl-indol-3-yl formamide was identified as a pathogen-inducible, tryptophan-derived compound whose biosynthesis is dependent on an intact PEN2 metabolic pathway. Based on the overaccumulation of 4-O-b-Dglucosyl-indol-3-yl formamide in the leaf tissues of pen3 mutants, it was proposed that its precursor is the cargo of PEN3, an ATP binding cassette transporter, in extracellular pathogen defense (Lu et al, 2015).…”