Identification and characterization of a serine protease from wheat leaves

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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Correspondence to Christof Rampitsch or Tim Xing.

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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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Keywords

  • Subtilisin-like protease
  • Programmed cell death
  • Stress
  • Wheat