Brassinin oxidase, a fungal detoxifying enzyme to overcome a plant defense – purification, characterization and inhibition

Abstract

Microbial plant pathogens display a variety of successful strategies to invade plant tissues and obtain the necessary nutrients that allow growth and reproduction. Plants fight back with no smaller a variety of weapons, including the synthesis of small to very large molecules to inhibit specific metabolic processes in the pathogen [1][2][3]. In general, plants under microbial attack produce de novo a blend of antimicrobial defenses known as phytoalexins, the specific components of which appear to depend on the type of stress [4,5]. Despite such an arsenal, fungal pathogens can disarm the plant by counterattacking with enzymes that detoxify promptly these phytoalexins [6][7][8]. The outcome of this 'arms race ' [3] frequently favors the pathogen, causing great crop devastation and substantial yield losses. Brassinin is a phytoalexin of great importance to crucifer plants, due to its dual role both as an antimicrobial defense and a biosynthetic precursor of several other phytoalexins. The toxophore group of brassinin is a dithiocarbamate, with an interesting resemblance to the potent fungicides used in the 1960s [9]. From a plant's perspective, it is highly desirable to prevent brassinin detoxification by any pathogen.Crucifers include a wide variety of crops cultivated across the world; for example, the oilseeds rapeseed and canola (Brassica napus and Brassica rapa) and vegetables such as cabbage (Brassica oleraceae var. capitata), cauliflower (Brassica oleraceae var. botrytis) or broccoli (Brassica oleraceae var. italica). In addition, both wild and cultivated crucifers are known to Blackleg fungi [Leptosphaeria maculans (asexual stage Phoma lingam) and Leptosphaeria biglobosa] are devastating plant pathogens with well-established stratagems to invade crucifers, including the production of enzymes that detoxify plant defenses such as phytoalexins. The significant roles of brassinin, both as a potent crucifer phytoalexin and a biosynthetic precursor of several other plant defenses, make it critical to plant fitness. Brassinin oxidase, a detoxifying enzyme produced by L. maculans both in vitro and in planta, catalyzes the detoxification of brassinin by the unusual oxidative transformation of a dithiocarbamate to an aldehyde. Purified brassinin oxidase has an apparent molecular mass of 57 kDa, is approximately 20% glycosylated, and accepts a wide range of cofactors, including quinones and flavins. Purified brassinin oxidase was used to screen a library of brassinin analogues and crucifer phytoalexins for potential inhibitory activity. Unexpectedly, it was determined that the crucifer phytoalexins camalexin and cyclobrassinin are competitive inhibitors of brassinin oxidase. This discovery suggests that camalexin could protect crucifers from attacks by L. maculans because camalexin is not metabolized by this pathogen and is a strong mycelial growth inhibitor.Abbreviations BO, brassinin oxidase; CKX, cytokinin oxidase ⁄ dehydrogenase; DEA, diethanolamine; FCC, flash column chromatography; PMS, phenazine methosulfate;...

Phytoalexins from Brassicaceae: News from the front

“…It has not been reported whether B. cinerea detoxifies camalexin (36). Finally, it is important to mention that a few other cruciferous phytoalexins were found to be inhibitors of BOLm, BHLmL2 and BHAb (Pedras et al, 2008a(Pedras et al, , 2009b. This very interesting discovery underscores the multiple ecological roles that phytoalexins have and explains why plants under stress biosynthesize complex blends.…”

“…Since the last reviews dealing with this aspect were published (Pedras and Ahiahonu, 2005;Pedras, 2008), three brassinin detoxifying enzymes from fungal pathogens were isolated and characterized. Brassinin oxidase (BOLm), catalyzing the detoxification of brassinin to indole-3-carboxaldehyde (65), was isolated from L. maculans virulent on canola (Pedras et al, 2008a), while brassinin hydrolases were isolated from L. maculans virulent on brown mustard (BHLmL2) and A. brassicicola (BHAb) (Pedras et al, 2009b). Since no dithiocarbamate oxidases or hydrolases appear to have been reported to date, these are new members of each of the enzyme groups.…”

Plant Antimicrobial Agents and Their Effects on Plant and Human Pathogens

“…This activity can be so important into the pathogenesis process that it can determine the disease severity of some fungi [90]. The discovering and understanding of inhibitors of phytoalexin-detoxifying enzymes is crucial to overcome this problem, and it opens a wide variety of biotechnological applications for a new generation of chemicals called paldoxins (from p hyto al exins d et ox ification in hibitor) designed to provide sustainable treatments of agricultural crops [91]. The use of paldoxin will allow the accumulation of the natural defence of the plant in an environmental safer way, since selective inhibitors are less likely to affect non-targeted organisms.…”

Plastic Transcriptomes Stabilize Immunity to Pathogen Diversity: The Jasmonic Acid and Salicylic Acid Networks within the Arabidopsis/Botrytis Pathosystem

“…Additionally, B. cinerea is a true haploid ascomycete that infects a wide range of evolutionarily distinct plant hosts, from bryophytes to eudicots. B. cinerea has elevated natural genetic variation that results in multiple major-effect polymorphisms in known virulence mechanisms, including the production of phytotoxic metabolites (Colmenares et al, 2002;Dalmais et al, 2011), enzymes that detoxify plant defense metabolites (Ferrari et al, 2003;Pedras et al, 2005Pedras et al, , 2007Pedras et al, , 2008Pedras et al, , 2009Pedras et al, , 2011Stefanato et al, 2009;Rowe et al, 2010), and the ability to degrade plant cell walls (Rowe and Kliebenstein, 2007;Schumacher et al, 2012Schumacher et al, , 2015Kumari et al, 2014). Because wild B. cinerea isolates have recombination and random mating, a population of isolates is a random intermixed sample of the diverse virulence mechanisms (Rowe and Kliebenstein, 2007;Kretschmer et al, 2009;Rowe et al, 2010;Kumari et al, 2014;Atwell et al, 2015;Corwin et al, 2016aCorwin et al, , 2016bZhang et al, 2016).…”