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First published online October 27, 2009; 10.1105/tpc.109.066167

The Plant Cell 21:3280-3295 (2009)
© 2009 American Society of Plant Biologists

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Transcript-Based Cloning of RRP46, a Regulator of rRNA Processing and R Gene–Independent Cell Death in Barley–Powdery Mildew Interactions[W],[OA]

Liu Xia,1, Matthew J. Moscoua,b, Yan Menga, Weihui Xua, Rico A. Caldoa,2, Miranda Shaverc,3, Dan Nettletond and Roger P. Wisea,b,c,e,4

a Department of Plant Pathology and Center for Plant Responses to Environmental Stresses, Iowa State University, Ames, Iowa 50011-1020
b Bioinformatics and Computational Biology, Iowa State University, Ames, Iowa 50011-3260
c National Institutes of Health–National Science Foundation Bioinformatics and Computational Systems Biology Summer Institute, Ames, Iowa 50011-1020
d Department of Statistics, Iowa State University, Ames, Iowa 50011-1210
e Corn Insects and Crop Genetics Research, U.S. Department of Agriculture–Agricultural Research Service, Iowa State University, Ames, Iowa 50011-1020

4 Address correspondence to rpwise{at}iastate.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. One-hundred 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Programmed cell death (PCD) is regulated by several diverse pathways in plants. Examples include the initiation and execution of senescence during plant development, the hypersensitive response (HR) during pathogen attack, and cellular damage sustained from various abiotic stresses (Lam, 2004Go). PCD can be initiated by internal or external factors and is under the control of active genetic programs. During leaf senescence, cell death usually starts from the tips or margins and progresses toward the base; however, earlier senescence can occur on targeted parts of the plant under environmental stress (Lim et al., 2007Go). In contrast with this cellular aging program, the HR is triggered when the plant recognizes a pathogen and generates a local cellular suicide, with the intent of halting pathogen progression (Sasabe et al., 2000Go). Each PCD program has discrete outcomes, and yet, conserved genes do exist among defense- and senescence-induced pathways (Quirino et al., 1999Go; Quirino et al., 2000Go). One such example is the tobacco (Nicotiana tabacum) harpin-induced1 gene, an HR cell death marker, which is also expressed at late stages of leaf senescence (Takahashi et al., 2004Go). Conversely, gene expression has also been found to be PCD specific, such as the induction of tobacco hsr203J for incompatible interactions with the bacterial pathogen Ralstonia solanacearum compared with the induction of Arabidopsis thaliana SAG12, encoding a putative Cys protease, during senescence (Pontier et al., 1999Go). Functional dissection of gene expression at key moments in cell death has facilitated our understanding of the mechanisms of cell-to-cell crosstalk and PCD as a whole.

In the interaction between host plants and their pathogens, cell death occurs in both incompatible and compatible responses. It is normally believed that, in the incompatible response, HR is activated by the R-AVR interaction, which restricts pathogen ingress, whereas compatibility triggered cell death may facilitate pathogen infection (Kim and Palukaitis, 1997Go; Greenberg et al., 2000Go). By contrast, several studies indicate that cell death and the prevention of pathogen invasion likely have separable functions in disease resistance (Bendahmane et al., 1999Go). For instance, the Rx gene conditions resistance to Potato virus X without cell death, but the Potato virus X coat protein elicits cell death when transformed into the Rx plants, leading to HR. Genetic investigations in oat (Avena sativa) revealed that Rds and Rih mediate HR, although they act independent of gene-for-gene resistance to the oat crown rust pathogen Puccinia coronata f. sp avenae (Yu et al., 2001Go). The Arabidopsis dnd1 and dnd2 mutants retain gene-for-gene mediated defense but with the reduced ability to produce HR (Yu et al., 1998Go, 2000Go). The dnd mutants also exhibit increased broad-spectrum resistance as a result of the activation of multiple defense pathways, none of which are required for the reduced HR (Genger et al., 2008Go). Hence, diverse pathways mediate cell death and each may have roles or control different aspects of plant–pathogen interactions.

Cellular features of PCD include fragmentation of nuclear DNA, signal transduction involving Ca2+ fluxes, changes in protein phosphorylation, increase in nuclear heterochromatin, and induction of reactive oxygen species (Greenberg, 1996Go; Pennell and Lamb, 1997Go). In plants, cell death is often correlated with development and metabolism and their associated differentiation, reproduction, and vegetative growth (Beers, 1997Go; Tamagnone et al., 1998Go). RNA processing represents an essential metabolic pathway throughout these stages. Central to this pathway is the exosome complex, which mediates 3' to 5' RNA processing and degradation in early pre-rRNA processing steps (Mitchell et al., 1997Go; Allmang et al., 2000Go). Recently, Chekanova et al. (2007)Go used genome-wide transcript mapping of exosome substrates to demonstrate the regulatory role of the exosome in mediating RNA quality control and stable structural RNA metabolism in plants. Other studies have shown that the exosome exhibits cell death–related nuclease function, leading to apoptotic DNA degradation in Caenorhabditis elegans and mammals (Parrish and Xue, 2006Go).

Barley (Hordeum vulgare) powdery mildew, caused by Blumeria graminis f. sp hordei (Bgh), is an ideal system to study the interactions between obligate fungal biotrophs with their hosts (Caldo et al., 2004Go). The well-characterized Bgh infection stages (Clark et al., 1993Go; Hall et al., 1999Go) provide the opportunity to monitor the host response and associated changes in gene expression upon pathogen attack. In this report, we isolated the fast neutron–derived, Bgh-induced tip cell death1 mutant, bcd1, and used Barley1 GeneChip expression profiles to discern the reprogramming of the transcriptome associated with the bcd1 mutation compared with its progenitor upon challenge by Bgh. Further bioinformatic and functional analytic methods were used to elucidate that RRP46, encoding a predicted rRNA processing protein, regulates cell death in an R gene–independent manner, implying a shared signaling pathway for rRNA processing and cell death, independent of the resistance response.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutant Characterization and Experimental Design
Mutants isolated for their pathogen-induced cell death provide an initial look at the cellular processes underlying crosstalk of cell death–mediated pathways (Lorrain et al., 2003Go; Love et al., 2008Go). In concert, the diversity of host responses to pathogen attack presents an opportunity to answer specific questions by selecting appropriate contrasts of plant and pathogen genotypes (Caldo et al., 2004Go). We used the bcd1 mutant, selected from a group of fast neutron–derived C.I. 16151 mutants (see Methods), where bcd1 was identified by the developing tip cell death upon Bgh inoculation while retaining resistant or susceptible responses to avirulent or virulent isolates, respectively (Figure 1 ). The bcd1 mutant exhibits rapid cell death in the top 2 to 3 cm of first leaves within 3 d after inoculation, even though Bgh conidia are attempting penetration at thousands of sites throughout its entire 10-cm length. To further characterize bcd1-mediated cell death, Bgh inoculated leaves were examined at 24, 48, 72, and 96 h after inoculation (HAI) using fluorescence microscopy. As shown in Figures 2A and 2B , Mla6-specified HR was observed in both wild-type C.I. 16151 and bcd1 leaves at 24 HAI. At 48 HAI, C.I. 16151 and bcd1 epidermal cells continued to exhibit HR (Figures 2C and 2D). In addition, bcd1 mesophyll cells displayed accumulation of phenolic compounds beneath epidermal cells that exhibit HR (Figure 2D), as well as in mesophyll below non-HR-producing epidermal cells (Figure 2E). At 72 HAI, bcd1 mesophyll exhibited more severe symptoms (Figures 2G and 2H), resulting in collapse at 96 HAI (Figure 2J). Pleiotropic effects are observed in bcd1, which manifest as macroscopic cell death on first and then second and third leaves of tillers prior to anthesis and a 23% increase in the number of sterile florets (P < 0.0001) compared with the wild type. Under normal greenhouse conditions (18 to 22°C, 16 h light), overall plant stature and tiller number are statistically equivalent; however, there is a trend toward a reduction for both in bcd1 mutants as plants near maturity. Additionally, cell death can occur in heat-shocked plants, suggesting a threshold model in the induction of this phenotype. As shown in Supplemental Table 1 online, bcd1 mutant plants treated at 37°C for six or more hours and returned to 24°C begin to exhibit cell death symptoms within 24 h, becoming more severe thereafter, whereas wild-type (C.I. 16151) plants were unaffected. By contrast, bcd1 mutant plants were identical to wild-type plants when treated at 4, 24 (control), or 30°C.


Figure 1
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  		Figure 1. Seedling Phenotypes and Experimental Design.

Transcript profiling was based on a split-split-plot design with replications as blocks, Bgh isolate as the whole-plot factor, plant genotype as the split-plot factor, and time as the split-split-plot factor. Seven-day-old plants (1st leaf) of the wild type (C.I. 16151) and fast neutron–derived mutant, bcd1, were inoculated with respective Bgh isolates 5874 (AVRa6) and K1 (avra6). Fifteen first leaves of inoculated barley seedlings were harvested at 0, 8, 16, 20, 24, and 32 HAI. One Barley1 GeneChip was used for each of the 72 split-split-plot experiment units corresponding to three replications x two isolates x two genotypes x six time points. The infection types shown above were photographed 7 d after inoculation. Infection type is classified on a scale of 0 to 4 and is shown in the top right of each panel. An infection type of 0 to 2 is resistant ("–" designates incompatible/no sporulation), whereas an infection type of 3 to 4 is susceptible ("+" designates compatible/abundant sporulation). 0, Immune; 1n, few small necrotic flecks (0.5 mm); 1 to 2n, significant small necrotic flecks (1 mm); 2N, abundant large necrotic flecks (1.5 mm); 4sp, completely susceptible; c, limited chlorosis; C, abundant chlorosis.

 

Figure 2
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  		Figure 2. Time-Course Fluorescence Microscopy of Wild-Type C.I. 16151 and bcd1 Mutant Seedlings in Response to Bgh 5874.

Seven-day-old C.I. 16151 and bcd1 seedlings were inoculated with Bgh 5874, and leaves were examined for evidence of HR by autofluorescence. Bars = 25 µm.

(A), (C), (F), and (I) Autofluorescence of C.I. 16151 epidermal cells at 24, 48, 72, and 96 HAI, respectively.

(B) Autofluorescence of bcd1 epidermal cells at 24 HAI.

(D) and (E) Accumulation of phenolic substances in the bcd1 mesophyll at 48 HAI.

(G) and (H) Local mesophyll cell collapse in bcd1 at 72 HAI.

(J) Collapse of bcd1 epidermal cells and mesophyll cells under the collapsed epidermal cells at 96 HAI.

 
To investigate the degree of interaction between PCD versus R gene–mediated resistance, our experimental design used two barley genotypes, C.I. 16151 (harboring the Mla6 powdery mildew resistance allele) and the bcd1 mutant described above, challenged with the Bgh isolates 5874 (AVRa6) and K1 (avra6), respectively (Figure 1). A time-course expression profile experiment was conducted based on a split-split-plot design described by Caldo et al. (2004)Go with replications as blocks, Bgh isolate as the whole-plot factor, plant genotype as the split-plot factor, and time as the split-split-plot factor. Fifteen first leaves of inoculated barley seedlings were harvested at 0, 8, 16, 20, 24, and 32 HAI. One Barley1 GeneChip (Close et al., 2004Go) was used for each of the 72 split-split-plot experiment units corresponding to three replications x two isolates x two genotypes x six time points. The resulting data set was then used for two purposes: (1) transcript-based isolation of the gene(s) responsible for the bcd1 phenotype, and (2) an assessment of cell death–mediated transcriptome reprogramming in both compatible and incompatible interactions in response to challenge with the biotrophic pathogen Bgh.

A Six-Gene Deletion Cosegregates with R Gene–Independent, bcd1-Mediated Tip Cell Death
It is known that fast-neutron mutagenesis generates deletions (Li et al., 2001Go); thus, we hypothesized that differential expression in the bcd1 mutant compared with the wild type could be caused by deletion of certain regulator(s), which control a coexpressed gene cluster associated with leaf tip cell death. Hence, to identify candidate genes that may be in such a deletion, we filtered the expression profiling database on two general criteria: (1) probe sets for which natural log expression in the bcd1 mutant was less than three through all six time points, indicative of a lack of measurable expression, and (2) probe sets for which expression was at least 4.5 times greater in wild-type C.I. 16151 than the bcd1 mutant at any one time point, indicative of a functional gene in wild-type plants, but not in the mutant. Twenty-two candidate genes were identified from this analysis (see Supplemental Table 2 online).

Deletion candidates were screened by both genomic PCR and RT-PCR. Six out of the 22 genes, represented by Barley1 probe sets Contig15422_at, Contig12722_s_at, Contig24342_at, Contig8225_at, Contig4201_s_at, and Contig9277_s_at, displayed no amplification in the bcd1 mutant in both assays and thus were concluded to be deleted (Table 1 , Figure 3C ). Two sets of genetic crosses were performed to test whether (1) the deleted genes cosegregated with the cell death phenotype and 2) bcd1-mediated cell death was genetically independent of R gene–mediated resistance. Specifically, the bcd1 mutant (Mla6/Mla6, bcd1/bcd1) was crossed to (1) the progenitor C.I. 16151 (Mla6/Mla6, Bcd1/Bcd1) and (2) cv Morex (mla/mla, Bcd1/Bcd1), respectively. Seven-day-old F2 seedlings were inoculated with Bgh 5874 and infection types were recorded another 7 d after inoculation. In cross 1, the ratio of the wild type versus plants displaying the tip cell death phenotype was 42:18, respectively, which fit the expected 3:1 ratio (Table 2 ). In the second test, 59 plants displayed resistance without cell death, nine plants showed resistance and cell death, 16 plants exhibited susceptibility without cell death, and three plants presented susceptibility and cell death, fitting a 9:3:3:1 ratio (Table 2). DNA was extracted from each plant in the two populations and used as template to amplify sequences representative of the six probe sets (Contig15422_at, Contig12722_s_at, Contig24342_at, Contig8225_at, Contig4201_s_at, and Contig9277_s_at). In both populations, except for Contig9277_s_at, no PCR-amplified products were observed from DNA of those plants displaying the cell death phenotype compared with those without cell death (Figure 4 ). As the genome sequence of barley is currently unavailable, we used Model Genome Interrogator at PLEXdb (http://www.plexdb.org/modules/MGI/) to derive the most probable genomic positions in rice (Oryza sativa). All five cosegregating candidate genes aligned to the region between Os03g63710 and Os03g63860 on rice chromosome 3, while the gene represented by Contig9277_s_at aligned to rice chromosome 5. Three additional genes were identified from the syntenic region on rice chromosome 3 (www.gramene.org) that were not on the Barley1 GeneChip. These sequences were used to identify their closest barley homologs and were subsequently tested for presence or absence via PCR amplification as described above. One of these genes (represented by GenBank accession number BF267800) was deleted and cosegregated with the cell death phenotype (Figure 3). Thus, a total of six genes were deleted in the bcd1 mutant and cosegregated the tip cell death phenotype. At the same time, results from cross 2 demonstrated that these six genes segregated independently from the Mla6 R gene, as plants homozygous for the deletion cosegregated with cell death regardless of the expected incompatible or compatible responses with Bgh isolate 5874 (AVRa6) (Figure 4B). Together with the autofluorescence experiments described above in the previous section, these results indicate that bcd1-mediated cell death is genetically separable from Mla6-specified HR.


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  		Table 1. Predicted Functions of Seven Deleted Genes in the bcd1 Mutant

 

Figure 3
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  		Figure 3. Integrated Map Depicting Chromosome 3 Region in Rice as Aligned with the Syntenous Barley Genetic Map, PCR Results, and Expression Data.

(A) Alignment of syntenous rice chromosome 3 region with barley positional orthologs on chromosome 5H. Barley genes were ordered and positioned according to the rice synteny (TIGR V5).

(B) Natural log expression difference of genes encompassing the deletion region between C.I. 16151 and mutant at 0, 8, 16, 20, 24, and 32 HAI according to the transcript accumulation data. Numbers across each line between (A) and (B) are Barley1 IDs for each probe set. Sequences with GenBank IDs are those not present on the GeneChip.

(C) Genomic-PCR and RT-PCR of deletion region genes, with wild-type C.I. 16151 on the left and mutant bcd1 on the right of each panel. Contig9277_s_at did not map to this deletion, but instead, chromosome 1HS.

 

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  		Table 2. Segregation and {chi}2 Analysis of F2 Populations

 

Figure 4
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  		Figure 4. Detection of Genes in the bcd1 Deletion in F2 Progeny.

The bcd1 mutant (Mla6/Mla6, bcd1/bcd1) was crossed to (1) wild-type C.I. 16151 (Mla6/Mla6, Bcd1/Bcd1) and (2) cv Morex (mla/mla, Bcd1/Bcd1), respectively. Seven-day-old F2 seedlings were inoculated with Bgh 5874, and infection types were recorded 7 d after inoculation. DNAs extracted from F2 populations of (A) C.I. 16151 (Mla6/Mla6, Bcd1/Bcd1) x bcd1 (Mla6/Mla6, bcd1/bcd1) and (B) Morex (mla/mla, Bcd1/Bcd1) x bcd1 were used as templates, respectively. PCR amplification was conducted to detect deletion candidate genes (Contig15422_at/CA031190, Contig12722_s_at, Contig24342_at, Contig8225_at, Contig4201_s_at, and BF267800). Except Contig9277_s_at, the other five genes were not amplified from plants exhibiting cell death but could be amplified from plants not exhibiting cell death. R, resistant plants; S, susceptible plants; C, cell death plants. Other deleted genes displayed identical results to Contig15422_at/CA031190 and Contig4201_s_at as pictured above. Contig9277_s_at, although deleted, does not cosegregate with cell death.

 
Genetic and Rice Synteny Mapping Positions the Deleted Region on Barley Chromosome 5H
We used 23,738 expression quantitative trait loci and 1596 transcript-derived markers from the Steptoe x Morex (St x Mx) doubled haploid mapping population (Potokina et al., 2008bGo, 2008aGo) to genetically map the deleted probe sets to barley chromosome 5H. The region encompassing the bcd1 deletion is highly syntenous with rice as described above, and the Barley1 probe sets identified (using tBLASTn) are ordered correctly within this region based on the transcript-derived marker and expression quantitative trait loci data (Figure 3). The proximal (left) border of the deletion is gene rich and permits a clear delineation of the breakpoint between Contig8931_at (LOC_Os03g63710) and Contig15422_at /CA031190 (LOC_Os03g63720). The distal (right) border is extremely gene poor, where most annotations of rice genes are retrotransposons of the classes Ty1-copia, Ty3-gypsy, a novel gene potentially encoding a protein with a WRKY domain, and several unclassified genes. The closest proximal gene that could be identified to the deletion breakpoint was Contig10531_at (LOC_Os03g63860) and was found to be present and expressed (Figure 3). Thus, we could predict the size of the deletion in rice to be between 30 and 70 kb, although barley is expected to have a much larger size due to repetitive elements.

Barley stripe mosaic virus–Induced Silencing of CA031190, Representing RRP46, Causes Tip Cell Death in Barley Leaves
Barley stripe mosaic virus (BSMV)-mediated virus-induced gene silencing (VIGS) was used to assess the functions of the six genes contained within the cosegregating bcd1 deletion (Table 1). Two primer sets per gene (see Methods) were used to amplify fragments for each of the six deleted genes from cDNA of wild-type C.I. 16151 (Bcd1/Bcd1) and inserted into BSMV-VIGS constructs according to Meng et al. (2009)Go. Seven-day-old C.I. 16151 plants were bombarded with each VIGS construct. Yellow-and-white stripe viral symptoms were observed in BSMV-infected leaves as the plants developed. Infected leaves were collected and processed to obtain recombinant virions, which were used to mechanically infect 7-d-old C.I. 16151 plants with at least eight seedlings in each treatment (see Methods). After 14 d, plants were inoculated with Bgh 5874, and phenotypes were assessed after an additional 7 d. Optimal silencing occurred at the third leaf stage, which was used for phenotypic observation (Meng et al., 2009Go).

As shown in Table 1, unigenes Hv21_15423 (represented by GenBank accession number CA031190) and Hv21_15422 (represented by Contig15422_at) both align to the genomic clone FJ652571. CA031190 represents residues 8 to 222 of the annotated open reading frame (ORF), predicted as RRP46 (see next section), while Hv21_15422 represents the 3' end of the ORF and 3' untranslated region. Hence, Contig15422_at was used for determination of transcript accumulation, while both CA031190 and Hv21_15422-derived sequences were used for BSMV-VIGS experiments (Figure 5A ). Plants infected with two constructs, BSMV:CA031190283 and BSMV:15422284, repeatedly developed tip cell death equivalent to the bcd1 mutant (Figure 5B). Tip cell death equivalent to the bcd1 mutant was not observed in the empty vector (BSMV:00) and mock or in experiments performed with the other five candidate gene constructs. Silencing experiments were repeated at least four times with each replicate exhibiting similar results. An additional three replications of 14 to 24 plants each were performed for BSMV:CA031190283 and BSMV:15422284 (example shown in Figure 5B). RNA gel blot analysis showed that the 1.2-kb RRP46 transcript was degraded in silenced plants with the cell death phenotype compared with the mock or BSMV:00 controls (Figure 5C). These results indicate that the RRP46-derived sequences within the cosegregating deletion in bcd1 mutant plants regulate Bgh-induced tip cell death.


Figure 5
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  		Figure 5. Silencing of CA031190 and Hv 15422 Causes Tip Cell Death.

(A) Exon/intron structure of RRP46 genomic clone (top) and cDNA (bottom), indicating positions of inserts for BSMV-VIGS constructs.

(B) Plants infected with two independent RRP46-derived constructs, BSMV:CA031190283 and BSMV:15422284, developed cell death equivalent to the bcd1 mutant after Bgh 5874 inoculation compared with the BSMV:00 (empty vector) and mock-infected plants. Each construct was used in six replicate experiments, of which at least eight plants per experiment were mechanically infected with each construct. The number above each group of leaves shows the number of plants with the pictured phenotype for replicate 6.

(C) RNA gel blot analysis showing the effect of BSMV-VIGS–mediated degradation on RRP46 transcript accumulation. RNA isolated from silenced 3rd leaves of mock (buffer control), BSMV:00, and BSMV:CA031190283 using a modified guanidium/acid-phenol protocol, size-fractionated through agarose gels, and hybridized with [{alpha}-P32]dCTP-labeled 15422284 insert. Transcripts were quantified by a phosphor imager and normalized using actin as a loading control. BSMV:CA031190283 silenced plants exhibited a 38% reduction in the quantity of RRP46 transcript compared with those treated with BSMV:00.

 
RRP46 Encodes a rRNA Processing Protein Conserved among Eukaryotes
To further investigate the gene structure from which CA031190 was transcribed, inverse and RT-PCR were used to recover 5', intron, and 3' sequences. The assembled sequences were further confirmed by amplification using primers flanking the CA031190 ORF using genomic DNA and cDNA template. The newly characterized sequence, Hv RRP46 (GenBank FJ652571), is comprised of seven exons (Figure 5A), the same number as the Os RRP46 (GenBank accession number AK069107) and Zm RRP46 (GenBank accession number EU964443) orthologs in rice and maize (Zea mays), respectively. The exon-intron borders are conserved as well. In barley, however, the introns are larger and mainly account for the difference in length between Hv RRP46 (>3.5 kb) and Zm RRP46 (2924 bp) or OsRRP46 (2562 bp). The predicted ORF of barley RRP46 is 720 bp, encoding a protein of 240 amino acids. A Pfam 22.0 (Finn et al., 2006Go) protein domain search of the Hv RRP46 ORF revealed significant similarity to RNase-PH (P value = 2.6e-42) and contains a single 3' exoribonuclease, phosphorolytic domain (Mitchell et al., 1997Go). The RNase-PH domain stretches from residues 14 to 134. A BLASTp search of the barley RRP46-encoded protein in The Institute for Genomic Research (TIGR) rice genome annotations (http://www.tigr.org/tdb/e2k1/osa1/) revealed 85% similarity to exosome complex exonuclease RRP46 (rRNA-processing protein 46), a protein involved in cytoplasmic rRNA processing and a component of the exosome 3' to 5' exonuclease complex (Allmang et al., 1999aGo). As illustrated in Figure 6 , a cross-taxa alignment was conducted using BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html), demonstrating the conservation of this protein and specific residues among eukaryotes.


Figure 6
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  		Figure 6. Multiple Sequence Alignment of 12 RRP46 Proteins.

Amino acid sequence alignment of 12 RRP46 proteins from plants (H. vulgare, O. sativa, Z. mays, and Arabidopsis), vertebrates (Homo sapiens and Mus musculus), insects (Anopheles gambiae and Drosophila melanogaster), nematode (C. elegans), yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe), and fungi (Neurospora crassa) was conducted using BioEdit (http://www.mbio.ncsu.edu/bioedit/bioedit.html). Sequences were derived from the available family information in the PANTHER database (www.pantherdb.org) (Thomas et al., 2003Go; Mi et al., 2007Go). The RNase-PH domain is positioned from amino acid residues 14 to 134. Amino acids highlighted in black are completely conserved, whereas those highlighted in gray contain weakly similar residues. Symbols: A colon indicates >50% observed for the most prevalent residue; a period indicates >75% observed for the most prevalent residue; and an asterisk indicates 100% observed for the most prevalent residue.

 
Constitutively Overexpressed Genes in the bcd1 Mutant are Associated with Ribosome Biosynthesis
Next, we used our time-course expression profiling data to investigate the effect of the RRP46 deletion on cell death–mediated transcriptome reprogramming in both compatible and incompatible interactions in response to challenge by Bgh. The statistical analysis was based on the overall pattern of expression over time, reflecting the kinetics of plant responses to pathogen infection (Caldo et al., 2004Go). Within this context, our primary strategy was focused on the identification of genes with time-course expression profiles that differed between the wild type and mutant. As summarized in Figure 7 (detailed data in Supplemental Data Sets 1 to 6 online), three contrasts were made to group genes associated with the bcd1-mediated cell death phenotype as opposed to R gene–mediated responses. The first contrast compared the time-course expression profile of the C.I. 16151 line (containing Mla6) to its fast neutron–derived mutant bcd1, both challenged with Bgh isolate 5874 (AVRa6). This comparison yielded those genes that were significantly differentially expressed between C.I. 16151 and bcd1 in response to the avirulent Bgh isolate 5874. Similarly, the second contrast was made between the time-course expression profiles of C.I. 16151 and bcd1 in response to the virulent Bgh isolate K1 (avra6). The third contrast took the average time-course expression profile in response to the two isolates for each genotype before contrasting C.I. 16151 with bcd1. The intersection of all three contrasts represent probe sets in which the time-course expression profile differs between the wild type and bcd1 mutant genotypes in a consistent manner for both isolates (Figure 7). A set of P values was obtained for each contrast as part of our mixed linear model analysis of the expression data as described in Methods. The intersection of the three contrasts contained 150 genes with a cutoff P value <0.0001 and an estimated false discovery rate (FDR) <0.6% (Nettleton et al., 2006Go). With the exception of 12 genes with variable patterns, 124 of these 150 genes were found to be constitutively overexpressed in the bcd1 mutant compared with C.I. 16151; while 14 genes displayed constitutively lower expression (Figure 8 ).


Figure 7
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  		Figure 7. Venn Diagram of Three Statistical Tests (Contrasts) to Delineate bcd1-Mediated Cell Death and R Gene–Mediated Incompatibility.

Totals of differentially expressed genes derived from mixed linear model analyses are shown in each cell. Contrasts were based on comparisons between C.I. 16151 (Bcd1) versus bcd1 mutant, each inoculated with Bgh isolates 5874 (AVRa6) or K1 (avra6). After specifying a P value cutoff of 0.0001, contrasts 1, 2, and 3 correspond to an FDR of 0.6, 0.2, and 0.09%, respectively. Individual cell summaries for P value, FDR, and natural log expression value per time point are provided in Supplemental Data Sets 1 to 6 online.

 

Figure 8
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  		Figure 8. Time-Course Expression Profiles of 150 Differentially Expressed Genes in C.I. 16151 and the Fast Neutron–Derived bcd1 Mutant.

(A) Standardized average signal intensities at each time point in wild-type C.I. 16151 and mutant inoculated with Bgh 5874 and K1 were used in the cluster analysis. A data matrix was constructed with genes in rows and time points of genotype-average isolate combinations in columns. Divisive clustering was used to measure similarities of transcript accumulation. Numbers 31, 43, 75, and 144 on the right-hand side of the heat map designate the relative position of expression profiles depicted in (B).

(B) Reciprocal expression profiles of representative overexpressed genes in the bcd1 mutant compared with its wild-type progenitor. The natural logarithm of signal intensities in C.I. 16151 and mutant with the average of the two Bgh isolates were plotted in graphs. Standard errors were calculated based on the mixed linear model used for analysis.

 
To view these differentially expressed genes in a biological context, Gene Ontology–based functional enrichment analysis was performed using EasyGO (Zhou and Su, 2007Go). Enrichment for genes associated with the ribosome was found in all three categories of the Gene Ontology (biological process, cellular component, and molecular function) (binomial test, P value = 7.4e-45). Cytoplasmic ribosomal proteins include both the large and small subunits, with no specific bias for either. In addition, predictive functional annotation for the 150 genes was performed by comparison to rice genome alignments, TIGR transcript assemblies, and the Dana-Farber Cancer Institute H. vulgare gene index via the PLEXdb probe set annotation tool (http://www.plexdb.org/modules/PD_general/tools.php) (see Supplemental Data Set 1 online). As shown in Table 3 , 68 of the 150 genes had annotations connected to the ribosome. This Gene Ontology and rice alignment-based overrepresentation suggests that the bcd1 mutation influences a regulatory pathway that controls the expression level of these genes (Barakat et al., 2001Go). Further investigation revealed that if we relaxed the FDR to 5%, all annotated ribosomal genes in barley were overexpressed in the bcd1 mutant. The remaining 26 genes, which had patterns other than constitutive overexpression, did not have significant overrepresentation in any category.


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  		Table 3. . Functional Categories of 150 Differentially Expressed Genes (bcd1 versus C.I. 16151) in Response to Challenge with Bgh Isolates 5874 and K1 (P Value < 0.0001).

 
Misprocessing of rRNAs Is Associated with Deletion of RRP46
Depletion of exosome components impacts RNA maturation and RNA quality (Houseley et al., 2006Go; Chekanova et al., 2007Go). In yeast, the depletion of Rrp46p protein increases the 3' extended forms of the 5.8S rRNA that extends to the length of the 7S pre-rRNA (Allmang et al., 1999aGo). To investigate the function of RRP46 in 5.8S rRNA maturation, RNA gel blot hybridization was performed to analyze the 3' extended forms of the 5.8S rRNA. The extended forms can be detected with a probe specific to internal transcribed spacers 2 (Mitchell et al., 1997Go). Seven-day-old wild-type C.I. 16151 and bcd1 mutant seedlings were challenged with Bgh 5874 and harvested at 0, 24, 48, 72, and 96 HAI for RNA extraction. As shown in Figure 9A , the bcd1 mutant rapidly accumulates 7S pre-rRNA and the 5.8S rRNA extended forms compared with the C.I. 16151 progenitor at all time points in both inoculated and noninoculated plants.


Figure 9
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  		Figure 9. Effects of RRP46 on rRNA Processing.

(A) RNA gel blot analysis of total RNAs extracted from 7-d-old seedlings of C.I. 16151 and bcd1 mutant. Paired flats were inoculated with Bgh isolate 5874 (AVRa6) or noninoculated and harvested at 0, 24, 48, 72, and 96 HAI. Five micrograms of total RNA per sample was fractionated on 8% polyacrylamide gels, and hybridization was performed with a probe specific to 3' extended forms for 5.8S rRNA (GenBank accession number Z11759.1). 5S and 5.8S rRNAs were visualized after ethidium bromide staining (bottom panel).

(B) Time-course expression profiling of polyadenylated 18S rRNA and 26S rRNA in C.I. 16151 and bcd1 mutant. The natural logarithms of signal intensities from PLEXdb accessions BB46 (this study), BB10 (Meng et al., 2009Go), and BB3 (Druka et al., 2006Go) were plotted in graphs. Standard errors were calculated based on the mixed linear model used for the analysis.

 
Recently, it has been reported that the depletion of exosome components cause aberrant accumulation of polyadenylated rRNA (Kuai et al., 2004Go; Chekanova et al., 2007Go; Lange et al., 2008Go). To further assess the role of RRP46 in rRNA processing, we analyzed the expression of polyadenylated 18S and 26S rRNA, represented by Barley1 probe sets Contig372_at and Contig711_at, respectively (Figure 9B). As shown in PLEXdb accession BB46 (this study), we found both polyadenylated rRNAs are significantly accumulated in bcd1 mutants compared with the wild type, in both compatible and incompatible interactions. Accumulation of these rRNAs was equivalent among inoculated and noninoculated wild-type plants (BB10) and within the range observed in 13 stages from seed to seed in barley development (BB3). Taken together, these data indicate that RRP46 in the bcd1 deletion is required for 5.8S rRNA maturation and also plays a role in the control of the level of polyadenylated rRNA.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Deletion of RRP46 Preconditions Barley Tip Cell Death
Contrasting cell death pathways have been found to have distinct and overlapping levels of interaction. Regardless of the path taken, the final destination is cellular suicide. Unlike lesion-mimic mutants that develop cell death constitutively, inoculation with Bgh induces tip cell death in bcd1 mutant plants; silencing of RRP46 contained within the bcd1 deletion generates an equivalent phenotype. Hence, data presented in this report demonstrate that RRP46 within the bcd1 deletion is required for R gene–independent PCD and the normal processing of rRNAs.

Physiological processes appear to be compromised by deficiencies caused by the bcd1 deletion, creating a cellular environment in which the plant is more vulnerable. Challenge by Bgh results in thousands of synchronous penetration attempts, which facilitates extensive collapse of both epidermal and mesophyll cells. This cellular collapse leads to accelerated leaf senescence, as senescence similarly begins from the tip or margins (Lim et al., 2007Go). A model of accelerated senescence does not account for the accompanying necrosis, which may be exacerbated by the underlying plant–pathogen interactions and resulting signal cascades. Thus, the absence of regulation by RRP46 may also lower the threshold for cell death in pathogen-attacked or presenescing cells (Shirasu and Schulze-Lefert, 2000Go).

During plant–pathogen interactions, cell death is believed to be associated with R gene–mediated defense and the ensuing HR (Greenberg and Yao, 2004Go). However, nonspecific induction of gene expression during the early stages of infection is not fundamentally different in incompatible and compatible barley–Bgh interactions (Boyd et al., 1995Go; Caldo et al., 2004Go; Caldo et al., 2006Go). Since bcd1-mediated cell death occurs in both incompatible and compatible responses (Figure 1), it is possible that the initiation of cell death may occur during these early pathogen infection stages. Interestingly, a GCC-box (GCCGCC) is found 10 times in the barley RRP46 promoter compared with 2.05% of all rice genes with 10 or more. The GCC-box is also found in other pathogen-responsive genes and is known to function as an ethylene- and jasmonate-responsive element (Lorenzo et al., 2003Go; Bouchez et al., 2007Go). Moreover, the GCC-box is the DNA target of ETHYLENE RESPONSE FACTOR1 (ERF1), which encodes a transcription factor that regulates the expression of pathogen response genes that prevent disease progression (Lorenzo et al., 2003Go). Hence, a possible scenario might involve the GCC-box as an RRP46 promoter element that is responsive to an ERF1-type transcription factor. In wild-type plants, RRP46 may potentiate a form of R gene–independent cell death, and without RRP46, plants succumb to the stress response generated by Bgh.

Destabilization of the rRNA/Protein Signaling Pathway
rRNA, the catalytic component of the ribosomes, accounts for ~80% of the total RNA in the eukaryotic cell (Kampers et al., 1996Go). The primary transcripts produced from most rRNA genes are extensively processed to yield the mature, functional forms (Lodish et al., 2000Go). Functional coordination of rRNAs and ribosomal proteins (r-proteins) assemble the ribosome, subsequently translating RNA into protein. Thus, preservation of steady state levels and cellular functions of ribosomes ensures normal metabolism in plants. However, in bcd1 mutant plants, genes encoding known constituents of ribosomes are constitutively expressed compared with its wild-type progenitor (see Supplemental Data Set 1 online; Figure 8). Since sufficient amounts of each r-protein must be produced to allow correct assembly and processing of rRNAs (Laferté et al., 2006Go; Michels and Hernandez, 2006Go), the misprocessing caused by the absence of a functional RRP46 would likely result in their overaccumulation (Figure 9). This, in turn, may activate a regulatory pathway that generates an overabundance of r-proteins as opposed to the normal steady state requirement. This destabilizes the rRNA/protein signaling pathway, compromising the plants' normal metabolism and physiological processes (Quirino et al., 1999Go; Chekanova et al., 2000Go; Stirpe and Battelli, 2006Go). Therefore, the overabundance of rRNA and protein alters the plants metabolism, impairing its defense system and facilitating cell death development upon pathogen attack.

Dual Function of RRP46 and Tip Cell Death
RRP46 belongs to the exosome, a conserved multiexonuclease complex that mediates RNA processing and degradation (Mitchell et al., 1997Go). The exosome core is characterized by a hexameric ring composed of six RNase PH domain-type proteins (RRP41, RRP42, RRP43, RRP45, RRP46, and MTR3) (Mian, 1997Go). In yeast and human, studies have shown that all the core subunits are vital to the normal activity of exosome to ensure its function (Allmang et al., 1999aGo, 1999bGo; Liu et al., 2006Go). Recently, Parrish and Xue (2003Go, 2006Go) described the degradeosome, an apoptotic DNA degradation pathway in C. elegans. One of its components, CRN-5, appears to be a functional homolog of RRP46. Silencing of crn-5 generates accumulation of TUNEL-reactive DNA intermediates in apoptotic cells, indicating its importance in apoptosis (Parrish and Xue, 2003Go). In our study, silencing of RRP46 resulted in a phenotype equivalent to the bcd1 mutant after challenge with Bgh. However, at this time, we have not observed evidence of bcd1-dependent DNA laddering (L. Xi and R.P. Wise, unpublished data), suggesting a lack of apoptosis in the generation of the cell death phenotype. More likely, the abundance of constitutively overexpressed ribosomal genes observed in the bcd1 mutant is consistent with the RNA processing function of RRP46. Thus, it is likely that without RRP46, the superfluous rRNA and ribosomal protein compromise the plants' normal metabolism and impairs the defense system, making them more susceptible to stress, resulting in cell death upon pathogen penetration. As cell death and overexpression of ribosomal-related genes were both observed in the bcd1 mutant, we hypothesize that bcd1-mediated cell death can be caused either by the direct cell death regulatory function of RRP46 or by the impaired pathways for metabolism and defense due to loss-of-regulation of rRNA/r-protein processing or by the interaction of these perturbed systems.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Fungal Isolates
Blumeria graminis f. sp hordei (Bgh) isolates 5874 (AVRa6) and K1 (avra6) were propagated on Hordeum vulgare cv Manchuria (C.I. 2330) in separate growth chambers at 18°C with 16 h of light and 8 h of darkness.

Isolation of Fast Neutron–Derived Mutant bcd1
The C.I. 16151 line was obtained by introgression of the Mla6 gene into the universal susceptible cv Manchuria (Moseman, 1972Go). Seeds of C.I. 16151 were treated with fast neutrons at 4 Gy Nf at the International Atomic Energy Agency, Vienna, Austria. M1 seeds were space planted at the USDA–Agricultural Research Service Small Grains Laboratory in Aberdeen, Idaho. Single spikes from each individual M1 plant were harvested to represent the M2 family, which was screened for mutant segregants by sowing intact spikes consisting of 25 to 40 seeds in potting soil following the method of Wise and Ellingboe (1985)Go. When the first leaves were completely unfolded (~10 cm high), plants were inoculated with Bgh isolate 5874 (AVRa6) and families were scored for infection type 7 d after inoculation. Families segregating three wild-type to one mutant for either cell death symptoms or sporulating Bgh colonies were identified as putative mutants. Mutant segregants were selected for rescue, advanced to the M3 generation, and then retested for homozygosity with Bgh 5874 as described by Meng et al. (2009)Go. Fast neutron–induced mutant m11542, harboring the bcd1 deletion, was confirmed as a single locus by genetic complementation, DNA gel blot (Halterman et al., 2001Go), and Barley1 GeneChip (Caldo et al., 2004Go, 2006Go) analyses.

Analysis of HR
First leaves were harvested at time points and fixed and cleared in fixation solution (ethanol:acetic acid = 3:1). Hypersensitive responses were examined using a fluorescence microscope (Leitz Fluovert) with a fluorescein filter set.

Microarray Experiment Design
Planting, stage of seedlings, harvesting, and experimental design were part of a larger experiment described by Caldo et al. (2004)Go. Briefly, C.I. 16151 (Mla6/Mla6, Bcd1/Bcd1) and the bcd1 mutant (Mla6/Mla6, bcd1/bcd1) were planted in separate 20 x 30-cm flats using sterilized potting soil. Each experimental flat consisted of six rows of 15 seedlings, with rows randomly assigned to one of six harvest time points (0, 8, 16, 20, 24, and 32 HAI). Seedlings grown to the 1st leaf stage with 2nd leaf unfolded were inoculated with a high density of fresh conidiospores (80 ± 20 spores/mm2). Groups of flats were placed at 18°C (8 h darkness, 16 h light) in separate controlled growth chambers corresponding to the Bgh isolates. Rows of plants were harvested into liquid nitrogen at the assigned time points. The entire experiment was repeated three times in a standard split-split-plot design with 72 experimental units (Kuehl, 2000Go).

Transcriptome Analysis
Total RNA was isolated using a hot (60°C) phenol/guanidine thiocyanate method as described by Caldo et al. (2004)Go. Probe synthesis, labeling and GeneChip hybridization, washing, staining, and scanning were performed at the Iowa State University GeneChip Core facility. Data processing and normalization were performed according to Caldo et al. (2004)Go. A gene-by-gene mixed linear model approach (Wolfinger et al., 2001Go) was employed to analyze the 22,792 probe sets on the Barley1 GeneChip using the SAS MIXED procedure. Each mixed linear model included genotype, isolate, time, and all their interactions as fixed effects. Random effects were included for replications, replication-by-isolate interactions (corresponding to whole-plot experimental units), replication-by-isolate-by-genotype interactions (corresponding to split-plot experimental units), and error terms (corresponding to split-split-plot experimental units). Three contrasts (described in Results) were tested for each gene as part of each mixed linear model analysis. For each contrast, the set of genes with P values < 0.0001 was estimated to have an FDR <0.7% using the histogram-based method described by Nettleton et al. (2006Go). The set of 150 genes with P values < 0.0001 for all three contrasts was identified for further analysis (see Supplemental Data Set 1 online). Gene Ontology enrichment was done using the EasyGO tool located at http://bioinformatics.cau.edu.cn/easygo/. Cluster analysis was performed using the cluster library in R. Average scaled signal intensities for the 150 genes were calculated from three replications and standardized; the dendrogram and heat map were generated using divisive clustering (diana) and heatmap functions in R, respectively.

Data Access
Detailed GeneChip data are publicly available at PLEXdb (http://www.plexdb.org/) under accession number BB46 and at the National Center for Biotechnology Information–Gene Expression Omnibus under accession number GSE14930. Complete cDNA and genomic clones of Hv RRP46 are annotated within GenBank under accession number FJ652571. Results of the analysis summarized in Figure 7 are provided as Supplemental Data Sets 1 to 6 online.

Genomic PCR and RT-PCR
RNA extraction of C.I. 16151 and the bcd1 mutant was performed as described above. For RT-PCR, single-strand cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen) using oligo (dT)20 as a primer. PCR was performed subsequently using Taq DNA polymerase, recombinant (Invitrogen) with specific primers according to each unigene sequence (Table 1; see Supplemental Table 2 online). PCR was also performed with 1 µg genomic DNA of the wild type and the mutant, respectively, using the same sets of primers. Sequences derived from probe sets corresponding to deletion borders (Contig8931_at and Contig10531_at) were used as positive controls (Figure 3; see Supplemental Table 2 online).

Cosegregation Analysis
bcd1 was crossed to the wild-type C.I. 16151 and Morex (C.I. 15773). Seven-day-old seedlings from each F2 population were inoculated with Bgh 5874. Infection phenotypes were scored 7 d after inoculation. A single leaf from each seedling was harvested for DNA extraction. PCR was performed using the population DNA as the template and primers representing the deletion gene candidates (see Supplemental Table 2 online). DNAs of C.I. 16151, the bcd1 mutant, Morex, and Manchuria were used as controls.

Isolation of genomic and cDNA Copies of Hv RRP46
All primers are listed in Supplemental Table 3 online. The 5' end of RRP46 was obtained by inverse PCR performed according to Meng et al. (2007)Go with minor modifications. One microgram of C.I. 16151 genomic DNA was subjected to overnight digestion with 5 units of PstI followed by self ligation. Subsequently, the self-ligation product was used as template for amplification with ThermalAce DNA polymerase (Invitrogen) using primers RRP46IPp1f and RRP46IPp1r. Reverse primer RRP46IPp1r was used for sequencing the PCR product.

RT-PCR was used to retrieve the 3' end of RRP46. Total RNA was extracted from C.I. 16151 plants according to the method of Caldo et al. (2004)Go. First-strand cDNA was synthesized using 2 µg of total RNA, oligo d(T)20 primer and Superscript reverse transcriptase III (Invitrogen) and subsequently used as the template to amplify the 3' end with AccuPrime High Fidelity Taq DNA Polymerase (Invitrogen) with the RRP46 gene-specific primer RRP46IPp1f and a poly-T primer with a linker sequence DTRAND1. One microliter of the resulting PCR product was used as template for a second round of PCR using the linker primer RAND1 and RRP46IPp1f. RRP46IPp1f was used for sequencing the PCR product. New 5' and 3' RRP46 primers (RRP46cdp1f and RRP46cdpr1) were designed from the resulting sequences and used to amplify complete cDNA and genomic clones, which are annotated within GenBank under accession number FJ652571.

BSMV Silencing Constructs
A DNA-based BSMV-VIGS system was used to silence candidate genes in wild-type C.I. 16151 (Meng et al., 2009Go). Total RNA was extracted from C.I. 16151 plants 20 HAI with Bgh 5874, according to the method of Caldo et al. (2004)Go. First-strand cDNA was synthesized using 2 µg of total RNA, oligo d(T)20 primer and Superscript reverse transcriptase III (Invitrogen). Primers were designed according to the RRP46 EST sequences, GenBank accession numbers CA031190 and CB881151 (Barley1 Contig15422_at), and are listed in Supplemental Table 3 online. First-strand cDNA was used as the template to amplify two independent EST sequence fragments of 283 and 284 bp, with introduced PacI and NotI recognition sites at the 5' and 3' ends, respectively. Amplified fragments were ligated in the antisense orientation into the PacI and NotI sites of the BSMV:{gamma} vector, with the resulting constructs designated as BSMV:CA031190283 and BSMV:15422284.

Mechanical Infection of BSMV and Powdery Mildew Inoculation
Seven to ten days after bombardment, plants displaying a BSMV infection phenotype (brown streak on the first leaf and chlorotic mosaics on the second leaf) were selected according to Meng et al. (2009)Go. Leaves from infected plants were ground with 2 to 5 volumes of 0.05 M phosphate buffer, pH 7.2, in an ice-cold mortar. Carborundum (0.05 g; Sigma-Aldrich) was added to the buffer for optimal grinding. Seven-day-old healthy barley seedlings were then infected with the appropriate recombinant virions by rubbing the first leaf with crude virus extract four to six times between thumb and index finger, with new gloves used for each construct to prevent contamination.

RNA Gel Blot Analysis
RNA was extracted from silenced plants using a modified guanidium/acid-phenol protocol. Eight micrograms of total RNA were denatured with DMSO for 1 h at 50°C, followed by size fractionation through a 1.2% Seakem GTG agarose (FMC) gel with 0.01 M iodoacetic acid to inhibit RNases. Electrophoresis was performed with 10 mM Na2HP04, pH 7, followed by transfer to Hybond XL (Amersham), baked at 80°C for 1 h, and cross-linked with 220 mJ of light emitted by 302 nm bulbs in a Stratalinker 2400 (Stratagene). Hybridization was performed as described (Wise et al., 1996Go). Actin was used as a loading control.

Analysis of RRP46 Function on 5.8S rRNA Maturation
C.I. 16151 and the bcd1 mutant were grown in separate 20 x 30-cm flats as described above, and rows of plants were harvested into liquid nitrogen at the assigned time points. RNA was prepared as described by Caldo et al. (2004)Go. Five micrograms of total RNA per sample was size fractionated on 8% polyacrylamide gels containing 8 M urea in 0.5x TBE. RNAs were electroblotted to Hybond-XL membranes (GE Healthcare). The blots were dried and UV cross-linked with a Stratagene 2400. Hybridization was performed in Church buffer (0.5 M NaPO4, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA) with 20 pmol end-labeled oligonucleotide 5'-AAGAGGGTGGTTGGGAGCGT-3'. The blots were washed twice with 1x SSPE-0.1% SDS at 42°C. Subsequently, the blots were exposed to a storage phosphor screens (GE Healthcare), and screens were scanned by a Typhoon scanner 9410 (GE Healthcare).

Accession Numbers
Complete cDNA and genomic clones of Hv RRP46 are annotated within GenBank accession number FJ652571. Additional cDNA sequences representing Hv RRP46 can be found under GenBank accessions numbers CA031190 and CB881151.

Supplemental Data
The following materials are available in the online version of this article.

Supplemental Table 1. Temperature Sensitivity Exhibited by the Barley bcd1 Mutant as Observed 3 d after Treatment.
Supplemental Table 2. PCR Primers for Candidate Deletion Genes
Supplemental Table 3. Primers Used for VIGS Constructs, RRP46 RNA Gel Blot Analysis, and Gene Cloning.
Supplemental Data Set 1. Summary of P Value, False Discovery Rate (q), and Log Expression Value: C.I. 16151 (Bcd1) versus Fast Neutron C.I. 16151 Mutant (bcd1); Intersection of Contrast 1, Contrast 2, and Contrast 3.
Supplemental Data Set 2. Summary of P Value, False Discovery Rate (q), and Log Expression Value: C.I. 16151 (Bcd1) versus Fast Neutron C.I. 16151 Mutant (bcd1); Contrast 1 – (Union of Contrast 2 and Contrast 3).
Supplemental Data Set 3. Summary of P Value, False Discovery Rate (q), and Log Expression Value: C.I. 16151 (Bcd1) versus Fast Neutron C.I. 16151 Mutant (bcd1); (Intersection of Contrast 1 and 3) – Contrast 2.
Supplemental Data Set 4. Summary of P Value, False Discovery Rate (q), and Log Expression Value: C.I. 16151 (Bcd1) versus Fast Neutron C.I. 16151 Mutant (bcd1); Contrast 2 – (Union of Contrast 1 and Contrast 3).
Supplemental Dataset 5. Summary of P Value, False Discovery Rate (q), and Log Expression Value: C.I. 16151 (Bcd1) versus Fast Neutron C.I. 16151 Mutant (bcd1); (Intersection of Contrast 2 and Contrast 3) – Contrast 1.
Supplemental Data Set 6. Summary of P Value, False Discovery Rate (q), and Log Expression Value: C.I. 16151 (Bcd1) versus Fast Neutron C.I. 16151 Mutant (bcd1); Contrast 3 – (Union of Contrast 1 and Contrast 2).


   Acknowledgments
 
The authors thank Liz Miller for technical assistance during the mutant isolation, and Greg Fuerst for excellent technical assistance throughout the project. This research was supported by the USDA Initiative for Future Agriculture and Food Systems Grant 2001-52100-11346, NIH-NSF Bioinformatics and Computational Systems Biology Summer Institute grant 06-08769, and National Science Foundation Plant Genome grant 05-00461. This article is a joint contribution of the Iowa Agriculture and Home Economics Experiment Station and the Corn Insects and Crop Genetics Research Unit, USDA-Agricultural Research Service. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.


   Footnotes
 
1 Current address: Baylor College of Medicine, Houston, TX 77030. Back

2 Current address: Monsanto Company, St. Louis, MO 63167. Back

3 Current address: STERIS Corporation, St. Louis, MO 63133-1029. Back

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Roger P. Wise (rpwise{at}iastate.edu).

[W] Online version contains Web-only data. Back

[OA] Open access articles can be viewed online without a subscription. Back

www.plantcell.org/cgi/doi/10.1105/tpc.109.066167

Received February 12, 2009; Revision received August 29, 2009. accepted October 2, 2009.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Allmang, C., Kufel, J., Chanfreau, G., Mitchell, P., Petfalski, E., and Tollervey, D. (1999a). Functions of the exosome in rRNA, snoRNA and snRNA synthesis. EMBO J. 18: 5399–5410.[CrossRef][Web of Science][Medline]

Allmang, C., Mitchell, P., Petfalski, E., and Tollervey, D. (2000). Degradation of ribosomal RNA precursors by the exosome. Nucleic Acids Res. 28: 1684–1691.[Abstract/Free Full Text]

Allmang, C., Petfalski, E., Podtelejnikov, A., Mann, M., Tollervey, D., and Mitchell, P. (1999b). The yeast exosome and human PM-Scl are related complexes of 3' -> 5' exonucleases. Genes Dev. 13: 2148–2158.[Abstract/Free Full Text]

Barakat, A., Szick-Miranda, K., Chang, I.-F., Guyot, R., Blanc, G., Cooke, R., Delseny, M., and Bailey-Serres, J. (2001). The organization of cytoplasmic ribosomal protein genes in the Arabidopsis genome. Plant Physiol. 127: 398–415.[Abstract/Free Full Text]

Beers, E.P. (1997). Programmed cell death during plant growth and development. Cell Death Differ. 4: 649–661.[CrossRef][Web of Science][Medline]

Bendahmane, A., Kanyuka, K., and Baulcombe, D.C. (1999). The Rx gene from potato controls separate virus resistance and cell death responses. Plant Cell 11: 781–792.[Abstract/Free Full Text]

Bouchez, O., Huard, C., Lorrain, S., Roby, D., and Balague, C. (2007). Ethylene is one of the key elements for cell death and defense response control in the Arabidopsis lesion mimic mutant vad1. Plant Physiol. 145: 465–477.[Abstract/Free Full Text]

Boyd, L.A., Smith, P.H., Foster, E.M., and Brown, J.K.M. (1995). The effects of allelic variation at the Mla resistance locus in barley on the early development of Erysiphe graminis f. sp. hordei and host responses. Plant J. 7: 959–968.[CrossRef][Web of Science]

Caldo, R.A., Nettleton, D., Peng, J., and Wise, R.P. (2006). Stage-specific suppression of basal defense discriminates barley plants containing fast- and delayed-acting Mla powdery mildew resistance alleles. Mol. Plant Microbe Interact. 19: 939–947.[CrossRef][Web of Science][Medline]

Caldo, R.A., Nettleton, D., and Wise, R.P. (2004). Interaction-dependent gene expression in Mla-specified response to barley powdery mildew. Plant Cell 16: 2514–2528.[Abstract/Free Full Text]

Chekanova, J.A., et al. (2007). Genome-wide high-resolution mapping of exosome substrates reveals hidden features in the Arabidopsis transcriptome. Cell 131: 1340–1353.[CrossRef][Web of Science][Medline]

Chekanova, J.A., Shaw, R.J., Wills, M.A., and Belostotsky, D.A. (2000). Poly(A) tail-dependent exonuclease AtRrp41p from Arabidopsis thaliana rescues 5.8 S rRNA processing and mRNA decay defects of the yeast ski6 mutant and is found in an exosome-sized complex in plant and yeast cells. J. Biol. Chem. 275: 33158–33166.[Abstract/Free Full Text]

Clark, T.A., Zeyen, R.J., Smith, A.G., Bushnell, W.R., Szabo, L.J., and Vance, C.P. (1993). Host response gene transcript accumulation in relation to visible cytological events during Erysiphe graminis attack in isogenic barley lines differing at the Ml-a locus. Physiol. Mol. Plant Pathol. 43: 283–295.[CrossRef]

Close, T.J., Wanamaker, S.I., Caldo, R.A., Turner, S.M., Ashlock, D.A., Dickerson, J.A., Wing, R.A., Muehlbauer, G.J., Kleinhofs, A., and Wise, R.P. (2004). A new resource for cereal genomics: 22K barley GeneChip comes of age. Plant Physiol. 134: 960–968.[Abstract/Free Full Text]

Druka, A., et al. (2006). An atlas of gene expression from seed to seed through barley development. Funct. Integr. Genomics 6: 202–211.[CrossRef][Medline]

Finn, R.D., et al. (2006). Pfam: Clans, web tools and services. Nucleic Acids Res. 34: D247–D251.[Abstract/Free Full Text]

Genger, R.K., Jurkowski, G.I., McDowell, J.M., Lu, H., Jung, H.W., Greenberg, J.T., and Bent, A.F. (2008). Signaling pathways that regulate the enhanced disease resistance of Arabidopsis "Defense, No Death" mutants. Mol. Plant Microbe Interact. 21: 1285–1296.[CrossRef][Web of Science][Medline]

Greenberg, J.T. (1996). Programmed cell death: A way of life for plants. Proc. Natl. Acad. Sci. USA 93: 12094–12097.[Abstract/Free Full Text]

Greenberg, J.T., Silverman, F.P., and Liang, H. (2000). Uncoupling salicylic acid-dependent cell death and defense-related responses from disease resistance in the Arabidopsis mutant acd5. Genetics 156: 341–350.[Web of Science][Medline]

Greenberg, J.T., and Yao, N. (2004). The role and regulation of programmed cell death in plant-pathogen interactions. Cell. Microbiol. 6: 201–211.[CrossRef][Web of Science][Medline]

Hall, A.A., Bindslev, L., Rouster, J., Rasmusses, S.W., Oliver, R.P., and Gurr, S.J. (1999). Involvement of cAMP and protein kinase A in conidial differentiation by Erysiphe graminis f. sp. hordei. Mol. Plant Microbe Interact. 12: 960–968.[CrossRef][Web of Science][Medline]

Halterman, D., Zhou, F., Wei, F., Wise, R.P., and Schulze-Lefert, P. (2001). The MLA6 coiled-coil, NBS-LRR protein confers AvrMla6-dependent resistance specificity to Blumeria graminis f. sp. hordei in barley and wheat. Plant J. 25: 335–348.[CrossRef][Web of Science][Medline]

Houseley, J., LaCava, J., and Tollervey, D. (2006). RNA-quality control by the exosome. Nat. Rev. Mol. Cell Biol. 7: 529–539.[CrossRef][Web of Science][Medline]

Kampers, T., Friedhoff, P., Biernat, J., Mandelkow, E.M., and Mandelkow, E. (1996). RNA stimulates aggregation of microtubule-associated protein tau into Alzheimer-like paired helical filaments. FEBS Lett. 399: 344–349.[CrossRef][Web of Science][Medline]

Kim, C.H., and Palukaitis, P. (1997). The plant defense response to cucumber mosaic virus in cowpea is elicited by the viral polymerase gene and affects virus accumulation in single cells. EMBO J. 16: 4060–4068.[CrossRef][Web of Science][Medline]

Kuai, L., Fang, F., Butler, J.S., and Sherman, F. (2004). Polyadenylation of rRNA in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 101: 8581–8586.[Abstract/Free Full Text]

Kuehl, R.O. (2000). Design of Experiments: Statistical Principles of Research Design and Analysis. (Pacific Grove, CA: Duxbury Press).

Laferté, A., Favry, E., Sentenac, A., Riva, M., Carles, C., and Chédin, S. (2006). The transcriptional activity of RNA polymerase I is a key determinant for the level of all ribosome components. Genes Dev. 20: 2030–2040.[Abstract/Free Full Text]

Lam, E. (2004). Controlled cell death, plant survival and development. Nat. Rev. Mol. Cell Biol. 5: 305–315.[CrossRef][Web of Science][Medline]

Lange, H., Holec, S., Cognat, V., Pieuchot, L., Le Ret, M., Canaday, J., and Gagliardi, D. (2008). Degradation of a polyadenylated rRNA maturation by-product involves one of the three RRP6-like proteins in Arabidopsis thaliana. Mol. Cell. Biol. 28: 3038–3044.[Abstract/Free Full Text]

Li, X., Song, Y., Century, K., Straight, S., Ronald, P., Dong, X., Lassner, M., and Zhang, Y. (2001). A fast neutron deletion mutagenesis-based reverse genetics system for plants. Plant J. 27: 235–242.[CrossRef][Web of Science][Medline]

Lim, P.O., Kim, H.J., and Nam, H.G. (2007). Leaf senescence. Annu. Rev. Plant Biol. 58: 115–136.[CrossRef][Medline]

Liu, Q., Greimann, J.C., and Lima, C.D. (2006). Reconstitution, activities, and structure of the eukaryotic RNA exosome. Cell 127: 1223–1237.[CrossRef][Web of Science][Medline]

Lodish, H., Berk, A., Zipursky, L.S., Matsudaira, P., Baltimore, D., and Darnell, J. (2000). Molecular Cell Biology. (New York: W.H. Freeman).

Lorenzo, O., Piqueras, R., Sanchez-Serrano, J.J., and Solano, R. (2003). ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense. Plant Cell 15: 165–178.[Abstract/Free Full Text]

Lorrain, S., Vailleau, F., Balagué, C., and Roby, D. (2003). Lesion mimic mutants: Keys for deciphering cell death and defense pathways in plants? Trends Plant Sci. 8: 263–271.[CrossRef][Web of Science][Medline]

Love, A.J., Milner, J.J., and Sadanandom, A. (2008). Timing is everything: regulatory overlap in plant cell death. Trends Plant Sci. 13: 589–595.[CrossRef][Web of Science][Medline]

Meng, Y., Moscou, M.J., and Wise, R.P. (2009). Blufensin1 negatively impacts basal defense in response to barley powdery mildew. Plant Physiol. 149: 271–285.[Abstract/Free Full Text]

Meng, Y., et al. (2007). A systematic analysis of T-DNA insertion events in Magnaporthe oryzae. Fungal Genet. Biol. 44: 1050–1064.[CrossRef][Web of Science][Medline]

Mi, H., Guo, N., Kejariwal, A., and Thomas, P.D. (2007). PANTHER version 6: Protein sequence and function evolution data with expanded representation of biological pathways. Nucleic Acids Res. 35: D247–D252.[Abstract/Free Full Text]

Mian, I.S. (1997). Comparative sequence analysis of ribonucleases HII, III, II PH and D. Nucleic Acids Res. 25: 3187–3195.[Abstract/Free Full Text]

Michels, A.A., and Hernandez, N. (2006). Does Pol I talk to Pol II? Coordination of RNA polymerases in ribosome biogenesis. Genes Dev. 20: 1982–1985.[Free Full Text]

Mitchell, P., Petfalski, E., Shevchenko, A., Mann, M., and Tollervey, D. (1997). The exosome: A conserved eukaryotic RNA processing complex containing multiple 3'->5' exoribonucleases. Cell 91: 457–466.[CrossRef][Web of Science][Medline]

Moseman, J.G. (1972). Isogenic barley lines for reaction to Erysiphe graminis f. sp. hordei. Crop Sci. 12: 681–682.[Abstract/Free Full Text]

Nettleton, D., Hwang, J.T.G., Caldo, R.A., and Wise, R.P. (2006). Estimating the number of true null hypotheses from a histogram of p values. J. Agr. Biol. Envir. St. 11: 337–356.[CrossRef]

Parrish, J.Z., and Xue, D. (2003). Functional genomic analysis of apoptotic DNA degradation in C. elegans. Mol. Cell 11: 987–996.[CrossRef][Web of Science][Medline]

Parrish, J.Z., and Xue, D. (2006). Cuts can kill: The roles of apoptotic nucleases in cell death and animal development. Chromosoma 115: 89–97.[CrossRef][Web of Science][Medline]

Pennell, R.I., and Lamb, C. (1997). Programmed cell death in plants. Plant Cell 9: 1157–1168.[CrossRef][Web of Science][Medline]

Pontier, D., Gan, S., Amasino, R.M., Roby, D., and Lam, E. (1999). Markers for hypersensitive response and senescence show distinct patterns of expression. Plant Mol. Biol. 39: 1243–1255.[CrossRef][Web of Science][Medline]

Potokina, E., Druka, A., Luo, Z., Moscou, M., Wise, R., Waugh, R., and Kearsey, M. (2008b). Tissue-dependent limited pleiotropy affects gene expression in barley. Plant J. 56: 287–296.[CrossRef][Web of Science][Medline]

Potokina, E., Druka, A., Luo, Z., Wise, R., Waugh, R., and Kearsey, M. (2008a). Gene expression quantitative trait locus analysis of 16,000 barley genes reveals a complex pattern of genome-wide transcriptional regulation. Plant J. 53: 90–101.[Web of Science][Medline]

Quirino, B.F., Noh, Y.S., Himelblau, E., and Amasino, R.M. (2000). Molecular aspects of leaf senescence. Trends Plant Sci. 5: 278–282.[CrossRef][Web of Science][Medline]

Quirino, B.F., Normanly, J., and Amasino, R.M. (1999). Diverse range of gene activity during Arabidopsis thaliana leaf senescence includes pathogen-independent induction of defense-related genes. Plant Mol. Biol. 40: 267–278.[CrossRef][Web of Science][Medline]

Sasabe, M., Takeuchi, K., Kamoun, S., Ichinose, Y., Govers, F., Toyoda, K., Shiraishi, T., and Yamada, T. (2000). Independent pathways leading to apoptotic cell death, oxidative burst and defense gene expression in response to elicitin in tobacco cell suspension culture. Eur. J. Biochem. 267: 5005–5013.[Web of Science][Medline]

Shirasu, K., and Schulze-Lefert, P. (2000). Regulators of cell death in disease resistance. Plant Mol. Biol. 44: 371–385.[CrossRef][Web of Science][Medline]

Stirpe, F., and Battelli, M.G. (2006). Ribosome-inactivating proteins: progress and problems. Cell. Mol. Life Sci. 63: 1850–1866.[CrossRef][Web of Science][Medline]

Takahashi, Y., Berberich, T., Yamashita, K., Uehara, Y., Miyazaki, A., and Kusano, T. (2004). Identification of tobacco HIN1 and two closely related genes as spermine-responsive genes and their differential expression during the Tobacco mosaic virus-induced hypersensitive response and during leaf- and flower-senescence. Plant Mol. Biol. 54: 613–622.[CrossRef][Web of Science][Medline]

Tamagnone, L., Merida, A., Stacey, N., Plaskitt, K., Parr, A., Chang, C.F., Lynn, D., Dow, J.M., Roberts, K., and Martin, C. (1998). Inhibition of phenolic acid metabolism results in precocious cell death and altered cell morphology in leaves of transgenic tobacco plants. Plant Cell 10: 1801–1816.[Abstract/Free Full Text]

Thomas, P.D., Campbell, M.J., Kejariwal, A., Mi, H., Karlak, B., Daverman, R., Diemer, K., Muruganujan, A., and Narechania, A. (2003). PANTHER: A library of protein families and subfamilies indexed by function. Genome Res. 13: 2129–2141.[Abstract/Free Full Text]

Wise, R.P., Dill, C.L., and Schnable, P.S. (1996). Mutator-induced mutations of the rf1 nuclear fertility restorer of T-cytoplasm maize alter the accumulation of T-urf13 mitochondrial transcripts. Genetics 143: 1383–1394.[Web of Science][Medline]

Wise, R.P., and Ellingboe, A.H. (1985). Fine structure and instability of the Ml-a locus in barley. Genetics 111: 113–130.[Web of Science][Medline]

Wolfinger, R.D., Gibson, G., Wolfinger, E.D., Bennett, L., Hamadeh, H., Bushel, P., Afshari, C., and Paules, R.S. (2001). Assessing gene significance from cDNA microarray expression data via mixed models. J. Comput. Biol. 8: 625–637.[CrossRef][Web of Science][Medline]

Yu, G.X., Braun, E., and Wise, R.P. (2001). Rds and Rih mediate hypersensitive cell death independent of gene-for-gene resistance to the oat crown rust pathogen Puccinia coronata f. sp. avenae. Mol. Plant Microbe Interact. 14: 1376–1383.[CrossRef][Web of Science][Medline]

Yu, I., Fengler, K.A., Clough, S.J., and Bent, A.F. (2000). Identification of Arabidopsis mutants exhibiting an altered hypersensitive response in gene-for-gene disease resistance. Mol. Plant Microbe Interact. 13: 277–286.[CrossRef][Web of Science][Medline]

Yu, I.C., Parker, J., and Bent, A.F. (1998). Gene-for-gene disease resistance without the hypersensitive response in Arabidopsis dnd1 mutant. Proc. Natl. Acad. Sci. USA 95: 7819–7824.[Abstract/Free Full Text]

Zhou, X., and Su, Z. (2007). EasyGO: Gene Ontology-based annotation and functional enrichment analysis tool for agronomical species. BMC Genomics 8: 246.[CrossRef][Medline]


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Loss of an Exosome Complex Component Potentiates R Gene-Independent Cell Death in Barley
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