| Summary: | The Bowman-Birk family of plant protease inhibitors are serine protease inhibitors that can
simultaneously inhibit trypsin and/or chymotrypsin. Unique features include an
unusually high thermal stability, tolerance towards low pH and resistance towards the
action of proteases. Bowman-Birk protease inhibitors have been implicated in various
biotic and abiotic stress conditions in plants. LRW222 was recently identified by means of
SSH in Puccinia triticina-infected wheat and was found to be homologous to Wali5, a
wound- and aluminium stress-induced Bowman-Birk type protease inhibitor. The aim of this
study was to determine the expression levels and role of the LRW222 protein in Pucciniainfected
wheat.
This study identified and validated stable reference genes for qPCR gene expression
analysis in rust-infected wheat. qPCR is the preferred method to quantify mRNA levels, but
requires validated reference genes for data normalisation. As prescribed by the MIQE
guidelines, the GeNorm-Plus algorithm was used to examine the expression stability of six
candidate reference genes in resistant Avocet Yr1 wheat infected with P. triticina, P.
striiformis and P. graminis f. sp. tritici respectively. These reference genes were used in the
gene expression analysis of LRW222. Results indicated induced expression in both resistant
and susceptible wheat, indicating its possible involvement in the early general defence
response of wheat. Co-immunoprecipitation and mass spectrometry was used in an attempt
to identify LRW222-interacting proteins to shed light on its specific role in plant defence.
Protein identification was inconclusive, but results indicated that under natural conditions,
LRW222 may exist as multimers in wheat.
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