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European Journal of Plant Pathology

, Volume 146, Issue 2, pp 293–304 | Cite as

Identification and characterization of a serine protease from wheat leaves

  • Tao Fan
  • Natalia V. Bykova
  • Christof Rampitsch
  • Tim Xing
Article

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.

Keywords

Subtilisin-like protease Programmed cell death Stress Wheat 

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

Notes

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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Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2016

Authors and Affiliations

  1. 1.Morden Research and Development CentreAgriculture and Agri-Food CanadaMordenCanada
  2. 2.Canadian Grain CommissionWinnipegCanada
  3. 3.Department of Biology and Institute of BiochemistryCarleton UniversityOttawaCanada

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