Abstract
A putative serine protease with a potential role in the plant biotic and abiotic stress response was purified from wheat leaf apoplastic fluid and partially characterized. Following two-dimensional electrophoresis a protein of Mr = 75 k and a pI of 4.2 to 4.5 was observed. This protein displayed in-gel protease activity and was specifically inhibited by phenylmethanesulfonyl fluoride and partially inhibited by Ca2+ and Zn2+, but not by E-64 or leupeptin. An internal tryptic fragment of 13 amino acids was identified by MALDI QqTOF MS/MS, and this peptide showed a high level of homology (85–100 % identity) to a highly conserved region of known plant subtilisin-like proteases. We demonstrated that the protease activity increased until a late stage of wheat leaf development and increased in response to heat shock. In both cases Rubisco large subunit was degraded with time. Protease activity was also increased during biotic stress. Leaves challenged with leaf rust (Puccinia triticina), showed an approximately three fold increase in protease activity during an incompatible interaction, compared to activity in mock-inoculated leaves and to leaves in a compatible leaf rust interaction. These results suggest that the expression of this serine protease could be involved in the defense response against both abiotic and biotic stresses.
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Abbreviations
- 2DE:
-
two-dimensional electrophoresis
- ETI:
-
effector triggered immunity
- HR:
-
hypersensitive response
- IEF:
-
isoelectric focusing
- MALDI:
-
matrix-assisted laser desorption/ionization
- MAMPs:
-
microbe-associated molecular patterns
- MS/MS:
-
tandem mass spectrometry
- MTI:
-
MAMP-triggered immunity
- PCD:
-
programmed cell death
- PRRs:
-
pattern recognition receptors
- RbcL:
-
Rubisco large subunit
- ROS:
-
reactive oxygen species
- PR:
-
pathogenesis-related
- SA:
-
salicylic acid
- TOF:
-
time-of-flight
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Acknowledgments
Thanks to Ken Standing and Werner Ens in University of Manitoba for generous access to their MALDI-QqTOF prototype mass spectrometer and to Brent McCallum, AAFC for providing the rust races. This work was funded by an internal grant from AAFC to TX and CR.
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Fan, T., Bykova, N.V., Rampitsch, C. et al. Identification and characterization of a serine protease from wheat leaves. Eur J Plant Pathol 146, 293–304 (2016). https://doi.org/10.1007/s10658-016-0914-x
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DOI: https://doi.org/10.1007/s10658-016-0914-x