Effects of exogenous compounds on laccase activity and expression of laccase gene family members of Colletotrichum gloeosporioides

Abstract:【Objective】Mango (Mangifera indica Linn.) is the second most important tropical fruit in China. Colletotrichum gloeosporioides is the main pathogen of mango anthracnose, a serious disease reducing fruit production. It has large host- range and wide distributions. The main way of infection of Colletotrichum spp. is direct invasion of the melanized appressorium to the host. Melanin could maintain the integrity of the appressorium cell wall, prevent the efflux of intracellular glycerol, promote the formation of high turgor in the appressorium, and help the fungus to invade the host. So, melanin is con-sidered to be a virulence factor for many plants pathogenic fungi. The intracellular melanin of Colletotrichum is mostly dihydroxynaphthalene (DHN) melanin, whose synthesis pathway is composed of a number of key enzymes with the laccase, the last enzyme, which plays a role in oxidative polymerization and catalyzes the synthesis of melanin. The laccase is a polyphenol oxidase containing copper ions, and widely distributed in plants, fungi, bacteria, lichens and insects. The laccases often exist in the form of gene superfamily and are considered as potential drug targets. In addition to being influenced by culture conditions, the expression of laccase genes is also regulated by a variety of exogenous substances such as metal cations, inorganic anions and organic substances, in a dose-response manner. In previous work, we cloned 13 laccase genes from C. gloeosporioides. In order to know the expression pattern of the laccase gene family, their roles in pathogenesis of C. gloeosporioides, and subsequently explore their possibility as potential target of fungicides or adjuvants, the influence of exogenous substances on the laccase activity, melanin production, appressorium formation and the expression of 13 laccase genes were analyzed in this paper.【Methods】The crude laccase was obtained by filtration and centrifugation from C. gloeosporioides cultures when the extracellular laccase activity reached the peak. The effects of 18 anions and cations and 9 organic substances on the extracellular laccase activity were tested using a laccase kit. On this basis, 4 representative exogenous substances were selected and their effects on the internal laccase activity, melanin production and appressorium formation rate were determined. The expression of 13 laccase genes after treatment of 4 representative exogenous substances was analyzed by qRT-PCR, respectively.【Results】The extracellular laccase secretion of C. gloeosporioides reached the peak10 days after being cultuted in liquid medium. Among 27 tested exogenous substances, 20 showed inhibitory effects on the extracellular laccase activity, including Fe^{2 +} , Fe^{3 +} , NH4 + , Na + , K+ , Cl- , SO₃ ^2- , CO3 2- , HCO3 - , SO4 2- , NO3 - , H2PO4 - , HPO4 2- , cysteine, resveratrol, p-aniline, p-hydroxybenzoic acid, dimethyl sulfoxide, cinnamic acid and vanillic acid. The inhibitory effect of Fe2+ , Fe3+ , SO3 2- and cysteine was particularly obvious, with an inhibition rate over 90%. Ca2 + at 1, 5 and 10 mmol· L^{- 1} all promoted laccase activity, while Mn2 + , Cu2 + , Zn2 + and Mg2 + promoted at 1 mmol · L^-1 , and inhibited at 5 and 10 mmol·L^-1 . Methyl 4-hydroxy-3-methoxycinnamate and pyrogallonic acid inhibited at 1 and 5 mmol·L^-1 , but significantly promoted at 10 mmol·L^-1 . For the representative exogenous substances, 1 mmol·L^{- 1} Cu2 + and Mn2 + promoted intracellular laccase activity and appressorium formation, and promotion of Cu2+ was more obviously than Mn2+ , with the relative laccase activity of 149%. 1 mmol·L^-1 SO3 ^2- and cysteine inhibited intracellular laccase activity and appressorium formation, with the relative laccase activity of 74% and 31%, respectively. For melanin synthesis, 1 mmol·L^-1 Cu2+ significantly promoted melanin production, the melanin content increased by 11.36% compared with the control, while Mn2+ , SO3 2- and cysteine had no significant effect. The qRT-PCR results showed that 1 mmol·L^-1 Cu2+ induced the expression of about 3/4 of laccase genes, but inhibited the expression of the Cglac11 and Cglac12, and had no effect on the expression of the Cglac3. Mn2 + , SO3 2- and cysteine promoted the expression of about 1/3 of laccase genes, inhibited the expression of about 23% of laccase genes, and did not affect the expression of the rest laccase genes. Four exogenous substances significantly promoted the expression of the Cglac2, and had no effect on the expression of Cglac3.【Conclusions】The different exogenous substances at different concentrations showed different effects on the extracellular laccase activity of C. gloeosporioides. Cu2+ , Mn2+ , SO3 2- and cysteine had different effects on intracellular enzyme activity, appressorium formation and melanin production, as well as the expression of 13 laccase genes. It was preliminarily speculated that laccase genes Cglac2, Cglac12 and Cglac13 would be closely related to the exogenous Cu2+ , Mn2+ , SO3 2- and cysteine.