Departamento de Bioquímica e Biologia, Molecular da Universidade Federal do Ceará, Campus do Pici, Cx. Postal 6033, Fortaleza, Ceará, CEP 60451-970, Brazil.
Planta. 2011 Jul;234(1):183-93. doi: 10.1007/s00425-011-1392-1. Epub 2011 Mar 11.
Proteins from latex of Calotropis procera (CpLP), Plumeria rubra (PrLP), Carica candamarcensis (P1G10) and Euphorbia tirucalli (EtLP) were tested for antifungal activity against phytopathogens. CpLP and P1G10 inhibited each fungi analyzed. PrLP and EtLP did not exert inhibition. CpLP and P1G10 exhibited preferential inhibitory activity towards R. solani (IC₅₀ = 20.7 and 25.3 µg/ml, respectively). The inhibitory activity was lost after heat treatment or proteolysis, providing evidence for the involvement of proteins in the inhibitory effect. Treatment of CpLP or P1G10 with Dithiothreitol improved both, the endogenous proteolytic activity and the antifungal properties. Conversely, pre-treatment of CpLP or P1G10 with iodoacetamide drastically reduced endogenous proteolytic activities and partially abrogated antifungal activity. Similar results were observed when spores were challenged to germinate in the presence of laticifer proteins. The purified cysteine proteinase CMS2MS2 from Carica candamarcensis latex or papain (E.C. 3.4.22.2), a cysteine proteinase from latex of Carica papaya L., but not trypsin (EC 3.4.21.4) or chymotrypsin (EC 3.4.21.1), two serine proteases, replicated the results obtained with CpLP or P1G10, thus restricting the antifungal property to latex plant cysteine proteinases. CpLP, CMS2MS2 and papain induced production of reactive oxygen species in spores of F. solani, suggesting that inhibition could be linked to oxidative stress. Proteome analysis of CpLP by 2-D electrophoresis and MALDI-TOF-TOF confirmed the existence of various pathogenic-related proteins such as chitinases, peroxidases and osmotins. The results support that laticifer proteins are part of plant defense repertoire against phytopathogenic fungi.
从 Calotropis procera（CpLP）、Plumeria rubra（PrLP）、Carica candamarcensis（P1G10）和 Euphorbia tirucalli（EtLP）的乳胶中提取的蛋白质进行了抗真菌活性测试，以对抗植物病原体。CpLP 和 P1G10 抑制了分析的每种真菌。PrLP 和 EtLP 没有发挥抑制作用。CpLP 和 P1G10 对 R. solani 表现出优先的抑制活性（IC₅₀ = 20.7 和 25.3 µg/ml）。热处理或蛋白水解后，抑制活性丧失，这为蛋白质参与抑制作用提供了证据。用二硫苏糖醇处理 CpLP 或 P1G10 可提高内源性蛋白水解活性和抗真菌特性。相反，用碘乙酰胺预处理 CpLP 或 P1G10 可大大降低内源性蛋白水解活性，并部分削弱抗真菌活性。当挑战孢子在乳管蛋白存在下发芽时，观察到类似的结果。从 Carica candamarcensis 乳胶中纯化的半胱氨酸蛋白酶 CMS2MS2 或木瓜蛋白酶（E.C. 3.4.22.2），一种来自 Carica papaya L. 乳胶的半胱氨酸蛋白酶，但不是胰蛋白酶（EC 3.4.21.4）或糜蛋白酶（EC 3.4.21.1），两种丝氨酸蛋白酶，复制了与 CpLP 或 P1G10 获得的结果，从而将抗真菌特性限制在植物乳管半胱氨酸蛋白酶中。CpLP、CMS2MS2 和木瓜蛋白酶诱导 F. solani 孢子产生活性氧，表明抑制可能与氧化应激有关。通过 2-D 电泳和 MALDI-TOF-TOF 对 CpLP 的蛋白质组分析证实了存在各种与致病相关的蛋白质，如几丁质酶、过氧化物酶和渗透素。结果支持乳管蛋白是植物防御机制的一部分，可抵御植物病原体真菌。
