In Planta Proteomics and Proteogenomics of the Biotrophic Barley Fungal Pathogen Blumeria graminis f. sp. hordei>

Abstract

To further our understanding of powdery mildew biology during infection, we undertook a systematic shotgun proteomics analysis of the obligate biotroph Blumeria graminis f. sp. hordei at different stages of development in the host. Moreover we used a proteogenomics approach to feed information into the annotation of the newly sequenced genome. We analyzed and compared the proteomes from three stages of development representing different functions during the plant-dependent vegetative life cycle of this fungus. We identified 441 proteins in ungerminated spores, 775 proteins in epiphytic sporulating hyphae, and 47 proteins from haustoria inside barley leaf epidermal cells and used the data to aid annotation of the B. graminis f. sp. hordei genome. We also compared the differences in the protein complement of these key stages. Although confirming some of the previously reported findings and models derived from the analysis of transcriptome dynamics, our results also suggest that the intracellular haustoria are subject to stress possibly as a result of the plant defense strategy, including the production of reactive oxygen species. In addition, a number of small haustorial proteins with a predicted N-terminal signal peptide for secretion were identified in infected tissues: these represent candidate effector proteins that may play a role in controlling host metabolism and immunity.

Host-Induced Gene Silencing in Barley Powdery Mildew Reveals a Class of Ribonuclease-Like Effectors

“…These candidate secreted effector proteins (CSEP) differ from previously identified B. graminis avirulence proteins AVR A10 and AVR K1 , which do not contain a predicted signal peptide (Ridout et al 2006;Sacristán et al 2009). The CSEP superfamily includes many of the genes and proteins previously identified as specifically expressed in haustoria ) and in infected epidermis (Bindschedler et al 2009(Bindschedler et al , 2011. One member of this superfamily, CSEP0055, has recently been shown to reduce HI when silenced by HIGS and interacts with barley pathogenesis-related protein PR17c and is, therefore, an experimentally validated effector (Zhang et al 2012).…”

“…These genes encode proteins we had previously identified as abundant in infected tissues (Bindschedler et al 2009(Bindschedler et al , 2011 and which showed the greatest levels of differential transcript accumulation in infection structures (Spanu et al 2010).…”

“…The majority of these encode proteins identified by mass spectrometry as associated specifically with haustoria (Bindschedler et al 2009(Bindschedler et al , 2011. Others encode small secreted proteins that were found to be highly upregulated in infected epidermis containing haustoria (Spanu et al 2010).…”

“…hordei has been driven by the identification of i) genes specifically upregulated during infection (Both et al 2005), ii) RNAs specifically associated with haustoria Spanu et al 2010), iii) proteins found specifically in haustoria (Bindschedler et al 2009(Bindschedler et al , 2011, iv) genes predicted to encode small secreted proteins with no evident homologs in other fungi by means of a bioinformatic survey of the B. graminis f. sp. hordei genome , and v) characterization of avirulence genes in the fungus that confer race-specific incompatibility on barley genotypes with corresponding resistance genes (Ridout et al 2006;Sacristán et al 2009).…”

The Barley Powdery Mildew Candidate Secreted Effector Protein CSEP0105 Inhibits the Chaperone Activity of a Small Heat Shock Protein

“…Some of them share a Y/F/WxC motif in the N-terminal part of the mature protein (Bindschedler et al, 2009(Bindschedler et al, , 2011Godfrey et al, 2010;Spanu et al, 2010;Pedersen et al, 2012). Only a few CSEPs have been studied and shown to play a role in virulence.…”