Transcript-Based Cloning ofRRP46, a Regulator of rRNA Processing andRGene–Independent Cell Death in Barley–Powdery Mildew Interactions  

Abstract

Programmed cell death (PCD) plays a pivotal role in plant development and defense. To investigate the interaction between PCD and R gene-mediated defense, we used the 22K Barley1 GeneChip to compare and contrast time-course expression profiles of Blumeria graminis f. sp hordei (Bgh) challenged barley (Hordeum vulgare) cultivar C.I. 16151 (harboring the Mla6 powdery mildew resistance allele) and its fast neutron-derived Bgh-induced tip cell death1 mutant, bcd1. Mixed linear model analysis identified genes associated with the cell death phenotype as opposed to R gene-mediated resistance. Onehundred fifty genes were found at the threshold P value < 0.0001 and a false discovery rate <0.6%. Of these, 124 were constitutively overexpressed in the bcd1 mutant. Gene Ontology and rice (Oryza sativa) alignment-based annotation indicated that 68 of the 124 overexpressed genes encode ribosomal proteins. A deletion harboring six genes on chromosome 5H cosegregates with bcd1-specified cell death and is associated with misprocessing of rRNAs but segregates independent of R gene-mediated resistance. Barley stripe mosaic virus-induced gene silencing of one of the six deleted genes, RRP46 (rRNA-processing protein 46), phenocopied bcd1-mediated tip cell death. These findings suggest that RRP46, a critical component of the exosome core, mediates RNA processing and degradation involved in cell death initiation as a result of attempted penetration by Bgh during the barley-powdery mildew interaction but is independent of gene-for-gene resistance.

Polyadenylation in RNA Degradation Processes in Plants

“…Interestingly, about 300 transcripts accumulated either only in the RRP4-depleted or only in the RRP41-depleted sample. This confirmed that individual subunits of the core complex affect specific substrates and have different contributions to the in vivo activity of the exosome, as has already been suggested by the distinct phenotype of the mutants (Chekanova et al 2007;Hooker et al 2007;Xi et al 2009). However, about 500 transcripts were upregulated in both samples and likely represent the common substrates of the exosome core complex (Chekanova et al 2007).…”

“…In plants, the situation appears to be different, and more complex: First, in contrast to yeast and human exosomes, the plant core subunit AtRRP41 has a phosphorolytic activity that is stimulated by short poly(A) tails, at least in vitro (Chekanova et al 2000). Second, downregulation of individual subunits of the plant core exosome results in differential plant phenotypes (Chekanova et al 2007;Hooker et al 2007;Xi et al 2009). Moreover, downregulation of the core subunits AtRRP41, AtRRP4, or AtCSL4 resulted in accumulation of different polyadenylated RNAs, indicating that each of the proteins functions in polyadenylation-assisted RNA decay, albeit affects a distinct set of substrates (Chekanova et al 2007).…”

Pathogenesis-associated transcriptional patterns in Triticeae

“…Likewise transcriptional patterns of homologous genes and a similar neighbourhood in co-expression networks in different species might also support the identification of gene orthologs in different species (Mutwil et al, 2008;Schreiber et al, 2009). Furthermore, micro-array analyses proved effective in transcript-based cloning in barley forward genetic approaches (Xi et al, 2009;Zhang et al, 2009).…”

The eukaryotic RNA exosome: Same scaffold but variable catalytic subunits

“…Additionally, it has been suggested that the plant exosome displays an even higher degree of structural and functional flexibility as particular subunits seem to be dispensable for viability and processing of certain subsets of substrates. 31,43 This is in contrast to the S. cerevisiae exosome, where all Exo-9 subunits are essential for viability, 20 but reminiscent of data from D. melanogaster that suggest the presence of various subcomplexes consisting only of a subset of the Exo-9 subunits and co-factors. 25,32,44 This situation is quite unexpected from evolutionary and structural viewpoints and thus requires further experimental validation.…”