Abstract
Ralstonia solanaceraum is the quarantine plant pathogenic bacterium that causes bacterial wilt in over 200 host plants, which include economically important crops such as potato, tomato, tobacco, banana, and ginger. Alternative biological methods of disease control that can be used in integrated pest management are extensively studied. In search of new proteins with antibacterial activity against R. solanacearum, we identified L-amino acid oxidases (LAOs) from fruiting bodies of Amanita phalloides (ApLAO) and Infundibulicybe geotropa (CgLAO). We describe an optimized isolation procedure for their biochemical characterization, and show that they are dimeric proteins with estimated monomer molecular masses of 72 and 66 kDa, respectively, with isoelectric point of pH 6.5. They have broad substrate specificities for hydrophobic and charged amino acids, with highest Km for L-Leu, and broad pH optima at pH 5 and pH 6, respectively. An enzyme with similar properties is also characterized from the mycelia of I. geotropa (CgmycLAO). Fractionated aqueous extracts of 15 species of mushrooms show that LAO activity against L-Leu correlates with antibacterial activity. We confirm that the LAO activities mediate the antibacterial actions of ApLAO, CgLAO, and CgmycLAO. Their antibacterial activities are greater against Gram-negative versus Gram-positive bacteria, with inhibition of growth rate, prolongation of lag-phase, and decreased endpoint biomass. In Gram-positive bacteria, they mainly prolong the lag phase. These in vitro antibacterial activities of CgLAO and CgmycLAO are confirmed in vivo in tomato plants, while ApLAO has no effect on disease progression in planta. Transmission electron microscopy shows morphological changes of R. solanacearum upon LAO treatments. Finally, broad specificity of the antibacterial activities of these purified LAOs were seen for in vitro screening against 14 phytopathogenic bacteria. Therefore, these fungal LAOs show great potential as new biological phytoprotective agents and show the fruiting bodies of higher fungi to be a valuable source of antimicrobials with unique features.
Highlights
The search for new antibacterial agents is especially important against plant pathogenic bacteria where there are no effective chemical or biological agents available for plant protection (Payne et al, 2007; Sahu et al, 2017)
L-amino acid oxidase (LAO) represent a major component of snake venoms, where they serve as toxins, which have been studied in great detail to date
Proteins that showed antibacterial activities against R. solanacearum were isolated from the fruiting bodies of A. phalloides and I. geotropa using a two-step procedure of gel filtration (Figure 1) and hydrophobic-interaction chromatography (Figure 2)
Summary
The search for new antibacterial agents is especially important against plant pathogenic bacteria where there are no effective chemical or biological agents available for plant protection (Payne et al, 2007; Sahu et al, 2017) One such plant pathogen is the quarantine bacterium Ralstonia solanacearum (Smith, 1896) Yabuuchi et al, 1996, which is the active agent for bacterial wilt in the plant family Solanaceae. LAOs represent a major component of snake venoms, where they serve as toxins, which have been studied in great detail to date These are flavin adenine dinucleotide or flavin mononucleotide binding proteins, with molecular masses from 50 to 300 kDa, and isoelectric points between pH 4.0 and 9.4. A few LAOs have been isolated from fungi, a recent screening of LAO activities in fungal fruiting bodies revealed that they represent a new rich and readily accessible source of versatile and robust enzymes with LAO activities (Žun et al, 2017)
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