Molecular Characterization of the Purine Degradation Pathway Genes ALA1 and URE1 of the Maize Anthracnose Fungus Colletotrichum graminicola Identified Urease as a Novel Target for Plant Disease Control

Abstract

Fungal pathogenicity is governed by environmental factors, with nitrogen playing a key role in triggering pathogenic development. Spores germinating on the plant cuticle are exposed to a nitrogen-free environment, and reprograming of nitrogen metabolism is required for bridging the time needed to gain access to the nitrogen sources of the host. Although degradation of endogenous purine bases efficiently generates ammonium and may allow the fungus to bridge the preinvasion nitrogen gap, the roles of the purine degradation pathway and of the key genes encoding allantoicase and urease are largely unknown in plant pathogenic fungi. To investigate the roles of the allantoicase and urease genes ALA1 and URE1 of the maize anthracnose fungus Colletotrichum graminicola in pathogenic development, we generated ALA1:eGFP and URE1:eGFP fusion strains as well as allantoicase- and urease-deficient mutants. Virulence assays, live cell, and differential interference contrast imaging, chemical complementation and employment of a urease inhibitor showed that the purine degradation genes ALA1 and URE1 are required for bridging nitrogen deficiency at early phases of the infection process and for full virulence. Application of the urease inhibitor acetohydroxamic acid did not only protect maize from C. graminicola infection, but also interfered with the infection process of the wheat powdery mildew fungus Blumeria graminis f. sp. tritici, the maize and broad bean rusts Puccinia sorghi and Uromyces viciae-fabae, and the potato late blight pathogen Phytophthora infestans. Our data strongly suggest that inhibition of the purine degradation pathway might represent a novel approach to control plant pathogenic fungi and oomycetes.

Natural Urease Inhibitors Reduce the Severity of Disease Symptoms, Dependent on the Lifestyle of the Pathogens

“…On leaves treated with 10 mM p-BQ, HQ, DDC, and iCPAA, the formation of appressoria was only slightly reduced (data not shown), but plant invasion and differentiation of haustoria were significantly inhibited (Figure 3C). Consistent with previous data [11], AHA also inhibited appressorial penetration and haustorium formation and effectively protected wheat leaves from B. graminis f. sp. tritici infection.…”

“…The pre-penetration infection structures of biotrophic and hemibiotrophic fungi develop in the absence of external nitrogen [15]. Therefore, biotrophs and hemibiotrophs critically depend on purine degradation and active urease, and urease inhibitors may therefore interfere with plant invasion by these pathogens [11]. In contrast, necrotrophic fungi secrete toxins and/or generate reactive oxygen species (ROS) before entering the plant [24] and killing host cells, and nitrogenous molecules may become available prior to invasion (Figure 1).…”

“…Agrobacterium tumefaciensmediated random mutagenesis experiments have identified the allantoicase gene of the maize anthracnose fungus Colletotrichum graminicola as a virulence factor [10] and suggested that the generation of urea and, subsequently, ammonium via the purine degradation pathway may be a prerequisite for full virulence. Indeed, urease-deficient mutants of this fungus showed virulence defects and supplementing the infection droplets with ammonium restored the virulence of the ∆ure1 deletion strains [11]. Moreover, the addition of the commercially available urease inhibitor acetohydroxamic acid (AHA), which is structurally related to urea but not hydrolysable by urease [10], reduced the virulence of the C. graminicola WT strain and strengthened the idea that chemical inhibitors of urease activity may represent a novel class of agents protecting plants from fungal disease [11].…”

“…Indeed, urease-deficient mutants of this fungus showed virulence defects and supplementing the infection droplets with ammonium restored the virulence of the ∆ure1 deletion strains [11]. Moreover, the addition of the commercially available urease inhibitor acetohydroxamic acid (AHA), which is structurally related to urea but not hydrolysable by urease [10], reduced the virulence of the C. graminicola WT strain and strengthened the idea that chemical inhibitors of urease activity may represent a novel class of agents protecting plants from fungal disease [11].…”

Purine metabolism in plant pathogenic fungi

“…In the hemibiotroph Colletotrichum graminicola , the maize anthracnose fungus, the purine degradation genes ALA1 and URE1 encoding ureases are required for appressorial penetration and full virulence. The urease inhibitor acetohydroxamic acid (AHA) significantly protects the host, and interferes the incasice process of hemibiotrophs and biotrophs fungi, and oomycetes ( Benatto Perino et al, 2020 ). The inhibition of purine catabolic pathway could be a choice for fungicide exploration and application to control diseases caused by plant fungal pathogens and oomycetes.…”