Abstract
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.
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Abbreviations
- BSA:
-
Bovine serum albumin
- CMS2MS2:
-
The cysteine proteinase purified of the latex of Carica candamarcensis
- DAB:
-
3,3′-Diaminobenzidine
- DTT:
-
Dithiothreitol
- IAA:
-
Iodoacetamide
- IC50 :
-
The protein concentration reducing growth to 50% of control values
- LP:
-
Laticifer proteins
- ROS:
-
Reactive oxygen species
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Acknowledgments
Biochemical, functional and applied studies of latex proteins were supported by grants from FUNCAP, CNPq, CAPES, RENORBIO and IFS (M.V.R.). Authors are in debit with Prof. José Tadeu Abreu de Oliveira which provided fungi sources for assays.
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Souza, D.P., Freitas, C.D.T., Pereira, D.A. et al. Laticifer proteins play a defensive role against hemibiotrophic and necrotrophic phytopathogens. Planta 234, 183–193 (2011). https://doi.org/10.1007/s00425-011-1392-1
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DOI: https://doi.org/10.1007/s00425-011-1392-1