RAR1 Positively Controls Steady State Levels of Barley MLA Resistance Proteins and Enables Sufficient MLA6 Accumulation for Effective Resistance

Epitope-Tagged MLA1 and MLA6 Are Functional

We generated various epitope-tagged Mla1 and Mla6 derivatives. The epitope tags were introduced as N- or C-terminal fusions into genomic clones of Mla1 and Mla6, each comprising ∼2-kb native 5′ and 1-kb 3′ regulatory sequences (Figure 1A). In addition, we generated C-terminal fusions with the 30-kD green fluorescent protein (GFP). To find out whether the resulting fusion proteins were able to trigger race-specific resistance upon recognition of cognate avirulence gene products (AVRMLA1 and AVRMLA6, respectively), we delivered the plasmid DNA constructs into barley leaf epidermal cells using particle bombardment and scored MLA1 or MLA6 activity in single cells after challenge with Bgh spores (Figure 1B; three component assay, Zhou et al., 2001). During compatible interactions, delivery of wild-type MLA1 or MLA6 constructs permitted invasive fungal growth of isolates A6 (AvrMla6) and K1 (AvrMla1), respectively, in ∼75% of transformed cells (percentage haustorium index). During incompatible interactions, delivery of Mla1 or Mla6 constructs reduced the haustorium index to 2 and 8%, respectively, which is comparable to previously published data (Zhou et al., 2001). N-terminal fusions of small c-myc or hemagglutinin (HA) epitopes rendered both MLA1 and MLA6 derivatives partially or fully nonfunctional (Figure 1B). By contrast, when the same epitope tags were fused to the C termini of MLA1 and MLA6, the fusion proteins exhibited race-specific resistance activity similar to the wild-type level. Likewise, MLA activities were only slightly impaired or not affected upon C-terminal fusions to myc-HA or triple HA (3xHA) tags, whereas both MLA1-TAP and MLA6-TAP (for tandem affinity purification tag) fusion proteins were essentially nonfunctional. Because C-terminal fusions with the large GFP reporter (30 kD) to MLA1 and MLA6 are functional, unknown TAP-tag specific features but not its large size (20 kD) must render the MLA-TAP fusion proteins inactive or unstable. Collectively the same epitope affected MLA1 and MLA6 activity to a similar degree, and C-terminal fusions retained resistance activities in a tag-dependent manner.

We generated transgenic barley lines expressing MLA1 and MLA6 C-terminal fusions to c-myc or HA epitopes for subsequent studies. A minimum of five independent single-copy insertion lines were generated for each construct using Agrobacterium tumefaciens–mediated transformation. Transgenic plants expressing the tagged MLA derivatives as well as lines expressing untagged Mla1 or Mla6 triggered effective race-specific resistance toward the cognate Bgh strains (K1 and A6, respectively). The observed infection phenotypes were indistinguishable from nontransgenic barley containing Mla1 or Mla6 (data not shown). Transgenic lines expressing the N-terminal myc-MLA1 fusion were found to be fully susceptible upon inoculation with the cognate AvrMla1 expressing K1 isolate. Taken together, the activities of MLA derivatives were comparable in transgenic plants and in the transient single-cell gene expression assay.

Tissue-Specific Distribution of MLA in Leaf Tissues Does Not Coincide with Its Physiological Site of Action

We examined the accumulation of epitope-tagged MLA1-HA fusion protein in various plant tissues including root, stem, the first leaf, and young (growing) as well as fully developed leaves of 6-week-old plants in homozygous transgenic lines (Figure 2A). Protein gel blot analysis of total protein extracts using α-HA antiserum detected a signal at ∼110 kD in all tissues tested, which is in accordance with the molecular mass predicted for MLA1 (Figure 2A). The presence of MLA1-HA in roots is remarkable because powdery mildew fungi are known to attack only aerial epidermal tissue. To examine a potential preferential accumulation of MLA in the epidermis, the physiological target tissue of powdery mildews, we collected abaxial leaf epidermis that can be readily peeled away from detached first leaves. Surprisingly, the presence of MLA1 could not be demonstrated in epidermal tissue by the protein gel blot detection system used in these experiments. We estimate that MLA1 accumulates to at least 20-fold lower levels in epidermal tissue as opposed to total leaf protein if equal protein amounts are compared (Figure 2B; see Discussion; data not shown). This distribution contrasts with ROR2 syntaxin, a component of broad-spectrum penetration resistance to Bgh, which is known to preferentially accumulate in the leaf epidermis (Figure 2B; Collins et al., 2003). These data suggest that the distribution of MLA1 in leaves does not correlate with its main physiologic site of action. Because MLA1 protein was not detectable in the epidermis, all following experiments were based on whole leaf protein extracts.

Soluble and Membrane-Associated MLA Pools

NB-LRR R proteins are predicted intracellular proteins, but this has only been experimentally tested to our knowledge in two cases using R gene constructs driven by native regulatory sequences (Boyes et al., 1998; Axtell and Staskawicz, 2003). We performed basic cellular fractionation experiments to identify compartments containing MLA. After removal of cell debris at 16,000g, MLA1-HA was found in both soluble and in 100,000g microsomal fractions at 150 mM near-physiological salt concentration (Figure 2C). The relative MLA abundance in these two fractions was strongly dependent on salt concentrations of the extraction buffer. At 200 and 500 mM NaCl, increasing amounts of soluble MLA were found, whereas decreasing levels were seen in the membrane-associated fractions. Unlike this, two other tested intracellular proteins, cytosolic HSP90 and SGT1, were found at all tested salt concentrations only in the soluble fraction. ROR2 syntaxin, an intracellular protein containing a predicted single tansmembrane helix, was preferentially recovered in the microsomal fractions. These data suggest the existence of soluble and membrane-associated MLA pools. Because the salt concentration of the extraction buffer also influenced the total amount of MLA extractable from leaf tissue samples such that at higher salt concentrations significantly more MLA was recovered from equal amounts of tissue (Figure 2C, crude extract), we modified our experimental setup to find out whether MLA can reversibly change its location from one pool to the other. Crude extracts prepared at 150 mM NaCl were readjusted after removal of cellular debris at 16,000g to higher or lower salt concentrations, allowed to equilibrate, and then separated into soluble and microsomal fractions (Figure 2D). At higher salt concentrations, more MLA was found in the soluble fraction, whereas at lower concentrations, increasing amounts associated with the microsomal fraction. These data indicate loose and reversible associations of soluble MLA with membrane fractions, possibly through ionic interactions.

MLA1 and MLA6 Steady State Levels Are Different

We compared the abundance of MLA1-HA relative to MLA6-HA in nonchallenged leaves (Figure 3A). MLA6-HA accumulated to about fourfold lower levels in comparison with MLA1-HA in each of four tested independent transgenic lines. Semiquantitative RT-PCR experiments were performed to detect potential differences in Mla1 and Mla6 gene expression and showed indistinguishable amounts of RT-PCR products (see below and Figure 6D). This is consistent with the fact that the 5′ regulatory sequences driving the expression of Mla1 and Mla6 are highly similar (95% identity in a 750-bp contiguous region immediately upstream of the transcription start site). The difference between Mla gene expression levels and protein accumulation suggests the existence of a posttranscriptional regulatory mechanism.

Mla R specificities to Bgh appear to be alleles of one of three highly sequence-diverged NB-LRR R gene homologs at the complex Mla locus (Wei et al., 2002; Shen et al., 2003; Halterman and Wise, 2004). This makes it impossible to generate lines expressing two or more homozygous Mla R specificities by crossings between naturally polymorphic Mla accessions. We crossed Mla1-myc and Mla6-HA expressing transgenic lines and selected homozygous F3 individuals coexpressing both or expressing each transgene separately. Inoculation experiments with powdery mildew isolates expressing AvrMla1 or AvrMla6 revealed efficient Mla1- and Mla6-specific resistance in lines coexpressing both transgenes, thereby demonstrating that two Mla R gene specificities can be functional in a single plant when both specificities were either heterozygous (F1) or homozygous (F3). Interestingly, the isoform-specific MLA abundance remained unchanged in F3 individuals coexpressing MLA1-HA and MLA6-myc in comparison with F3 individuals expressing either transgene alone (Figure 3B). This demonstrates lack of potential allelic interference and shows that the isoform-specific accumulation differences are attributable to intrinsic properties of MLA1 and MLA6.

Rar1 Is Involved in MLA1 and MLA6 Abundance in Nonchallenged Plants

Transgenic barley plants expressing epitope-tagged Mla1-HA or Mla6-HA were crossed with a rar1-2 mutant line lacking detectable RAR1 protein (Freialdenhoven et al., 1994). F2 seedlings were selected containing each at least one Mla transgene copy and homozygous wild-type or mutant rar1 alleles. Analysis of F3 families derived from F2 individuals after selfing enabled us to identify plants that were homozygous at both the transgene and the Rar1 locus. Lines containing Mla1-HA triggered an effective AvrMla1-dependent immune response without macroscopically visible HR irrespective of the presence or absence of wild-type Rar1 (Figure 4F). Unlike this, effective Mla6-HA triggered resistance was clearly impaired in a rar1 mutant background upon challenge with the AvrMla6 containing Bgh isolate. In MLA6-HA rar1 plants, frequent necrotic patches were visible underneath fungal colonies, and the colony size was smaller compared with fully compatible interactions, which is indicative of ineffective resistance lagging behind the fungal infection process (trailing HR; data not shown). This differential impairment of Mla1 and Mla6 resistance in the rar1-2 mutant background is consistent with previous studies using nontransgenic barley (Jørgensen, 1996). Unexpectedly, we found a fourfold reduction of both MLA1-HA and MLA6-HA steady state levels in rar1 mutant backgrounds in nonchallenged plants (Figures 4A and 4D). This is surprising to us because previous genetic studies implied that RAR1 is not involved in the MLA1 resistance pathway (Jørgensen, 1996; Zhou et al., 2001). The decrease of MLA abundance in rar1 backgrounds is not accompanied by a reduction of Mla gene expression (Figure 4B; Mla1-HA not shown).

We also identified F3 individuals that were homozygous for MLA6-HA and contained one Rar1 wild-type copy (Rar1 rar1 plants). In these heterozygous Rar1 rar1 lines, MLA6-HA accumulation was indistinguishable in comparison with homozygous Rar1 lines, revealing that Rar1 gene dosage is not important for the control of MLA6 accumulation (Figures 4C and 4E). This is consistent with indistinguishable resistant infection phenotypes found in homozygous Mla6-HA containing lines differing for the presence of one or two Rar1 copies (data not shown).

In contrast with Rar1 gene dosage insensitivity, Mla1 copy number does affect the resistance phenotype. However, Mla1 dosage sensitivity can be detected macroscopically only in the absence of RAR1 (Figure 4F). Plants containing two Mla1 copies are fully resistant in a rar1 mutant background, whereas one Mla1 dosage in this background confers attenuated resistance leading to large necrotic patches beneath small fungal colonies as well as permitting occasional conidiospore formation (indicative of a trailing HR; Figure 4F). This demonstrates that Mla1 gene dosage becomes rate limiting for resistance in the absence of Rar1. Consistent with this, MLA1-HA abundance in transgenic plants carrying a single transgene copy were about half the level seen in plants containing two Mla1-HA copies, in both Rar1 and rar1 backgrounds (data not shown). We conclude that Mla1 functions independently of Rar1 only where MLA1 abundance exceeds a threshold level.

RAR1 May Specifically Control the Abundance of NB-LRR–Type R Proteins

To find out whether RAR1 interferes with the accumulation of other NB-LRR R proteins than MLA, we tested the abundance of Rx in N. benthamiana in the presence and absence of RAR1. Rx is a CC-NB-LRR type R protein that confers resistance to the potato virus X (PVX) and requires SGT1 for its function (Peart et al., 2002). We used transgenic N. benthamiana lines expressing Rx-HA under the control of native regulatory sequences (Lu et al., 2003). Gene silencing of RAR1 by virus-induced gene silencing strongly reduced both RAR1 and Rx abundance (Figure 5A). However, silencing of RAR1 did not impair the resistance response to PVX (data not shown). This demonstrates that the residual Rx protein in RAR1-silenced plants is still sufficient to trigger an effective resistance response.

To find out whether RAR1 elevates protein abundance of all intracellular LRR containing proteins, we examined the Arabidopsis F-box protein COI1, containing C-terminal LRRs, in the wild type, rar1, sgt1b, and the double mutant. COI1 is known to play an essential role in the signaling of jasmonate-dependent resistance responses (Xie et al., 1998). Unaltered COI1 steady state levels in wild-type and mutant backgrounds (Figure 5B) might indicate that RAR1 selectively interferes with NB-LRR R protein abundance.

MLA Abundance Is Inversely Correlated to RAR1 Requirement for Resistance

To obtain further insights into the link between RAR1 and MLA abundance in nonchallenged plants, we used two barley MLA chimeras, designated 11666 and 11166, whose functionality was previously examined by transient single-cell gene expression studies using the strong ubiquitin promoter (Shen et al., 2003). The CC and NB domains of these two chimeras are derived from MLA1, the LRRs are hybrids between MLA1 and MLA6, and the CT region is derived from MLA6 (Figure 6A). Both MLA chimeras confer AvrMla6-dependent resistance but differ in their requirement for Rar1 (resistance triggered by 11666 but not 11166 needs the presence of Rar1; 11666-mediated resistance is disrupted in the absence of Rar1, whereas wild-type Mla6 plants show intermediate resistance in rar1 mutants with a haustorium index of 40%; Shen et al., 2003). We generated transgenic barley containing HA epitope-tagged genomic clones of both MLA chimeras whose expression is driven by native Mla6 regulatory sequences. Wild-type Rar1 plants expressing chimera 11166-HA exhibited AvrMla6-dependent resistance (Figure 6E). The RAR1-dependent chimera 11666-HA permitted limited growth of the isolate A6 expressing AvrMla6 and unrestricted growth of the isolate K1 lacking AvrMla6, suggesting activation of an inefficient AvrMla6-triggered resistance. Necrotic patches beneath A6 colonies were indicative of a trailing HR and were absent beneath K1 colonies (Figure 6E). Protein gel blot analysis of leaf protein extracts derived from multiple independent single-copy transgenic lines expressing chimeras 11666-HA or 11166-HA revealed markedly higher steady state levels of the RAR1-independent 11166-HA chimera (Figure 6B). The abundance of chimera 11166-HA was found to be indistinguishable from wild-type MLA1-HA–expressing lines, whereas the 11666-HA protein was barely detectable (Figures 6B and 6C). These dramatic differences in chimera abundance are not the result of differential Mla gene expression levels (Figure 6D). The findings suggest an inverse correlation between MLA abundance and RAR1 requirement for resistance. It seems possible that AvrMla6-dependent 11166 resistance functions in the absence of RAR1 because the chimera accumulates to a level that is indistinguishable from wild-type MLA1. Conversely, chimera 11666-triggered resistance might exhibit enhanced dependence on RAR1 relative to wild-type MLA6 because of the diminished in planta accumulation.

MLA Protein Abundance Is Temperature Sensitive

We accidentally discovered that MLA proteins had disappeared in leaves harvested during hot summer days. To examine a potential temperature-sensitive MLA accumulation, we grew Mla1-HA and MLA6-HA transgenic seedlings for 7 d at 18°C and transferred them subsequently to 37°C (which is still below a typical heat shock for barley plants). We observed a marked and rapid decrease of both MLA1 and MLA6 abundance upon elevation of the ambient temperature. Interestingly, the reduction of MLA6 abundance was faster (detectable within 30 min) than of MLA1 (2 h), and both proteins remained at low levels at the end of the 8 h interval tested (Figure 7). Unlike this, exposure of the seedlings to the elevated temperature had no significant effect on RAR1, SGT1, and cytosolic HSP90 abundance, suggesting that MLA proteins might be intrinsically temperature sensitive. Unfortunately, we are unable to test whether the reduction of MLA abundance at elevated temperature impairs resistance to B. graminis because the fungus is unable to colonize barley at temperatures above 30°C (Aust, 1974; Jenkyn and Bainbridge, 1978).

MLA Isoform-Dependent Interactions with HvSGT1 and Isoform-Independent Interactions with HvHSP90

We searched for proteins that interact with MLA1 and MLA6 using the yeast two-hybrid method (Gyuris et al., 1993). For this purpose, we employed various domains or full-length MLA1 and MLA6 as baits and screened a barley prey cDNA library derived from leaf epidermal tissue (see Methods). We included in these screens baits comprising the N-terminal 46 amino acids (designated CC), the CC and nucleotide binding motifs (designated CC-NB), only the NB motifs (NB), or the LRRs and the C terminus (LRR-CT, Figure 8A). The most frequent MLA interactor identified in the prey library was barley SGT1 (26 independently isolated clones). This interaction was restricted to the MLA1-derived bait encompassing the LRR-CT region. The identified prey clones encoded either the complete coding sequence of SGT1 or three different truncations in the TPR domain, as well as one truncation immediately N-terminal to the SGS domain (Figure 8B). No prey clones were recovered encoding the CS plus SGS domains alone.

All MLA baits were tested in parallel in a directed screen for interactions with RAR1, SGT1, and cytosolic HSP90 together with further baits expressing the LRR-CT portions of MLA chimeras 11166 and 11666. No association was detected between any tested MLA bait and barley RAR1. The LRR-CT portion of MLA1 and 11166, but not of MLA6 and 11666, was found to interact with full-length barley SGT1 as well as with both full-length Arabidopsis SGT1 isoforms, designated AtSGT1a and AtSGT1b (Figure 8C; Austin et al., 2002; Azevedo et al., 2002). By contrast, cytosolic HvHSP90 interacted with the LRR-CT of all tested MLA variants. Baits encoding full-length MLA1 or MLA6 did not interact with either SGT1 or HSP90, although the LexA fusion baits were detectable in yeast cells (data not shown). This might be indicative of inhibitory intramolecular MLA domain interactions (see Discussion).

Subcellular Localization and Tissue-Specific Abundance of MLA Proteins Might Be Governed by Interacting Partners

Little is known about the subcellular localization of predicted intracellular NB-LRR R proteins expressed at native levels. Detectable Arabidopsis RPS2 was found to be entirely plasma membrane associated as well as the majority of Arabidopsis RPM1 (Boyes et al., 1998; Axtell and Staskawicz, 2003). Unlike this, at near-physiological salt concentrations, the bulk of MLA appears soluble, and a second pool associates with membranes (Figures 2C and 2D). Because the association with microsomal membrane fractions was reversible and dependent on ionic strength, the subcellular localization of MLA might be governed by charge interactions with integral or peripheral membrane proteins. For example, the RPM1 and RPS2 interactor RIN4 colocalizes with these two NB-LRR proteins at the plasma membrane (Axtell and Staskawicz, 2003; Mackey et al., 2003). RIN4 was also shown to be required for the accumulation of RPM1. Thus, it is conceivable that the tissue-specific difference in MLA abundance seen in epidermis and total leaf reflects an unequal tissue distribution of an MLA1 interactor.

The presence of high MLA levels in total leaf and undetectable MLA protein in the epidermis is counterintuitive because host cell invasion by powdery mildew fungi is restricted to epidermal cells (Figure 2B). Because epidermal cell monolayers prepared from partially dissected coleoptiles of Mla1 genotypes were shown to mount a race-specific resistance response (including single cell HR; Bushnell and Liu, 1994; Schiffer et al., 1997), epidermal cells must be competent to detect the cognate fungal effector and thus contain presumably very low levels of MLA1.

A Universal Role for RAR1 in Folding Preactivated NB-LRR–Type R Proteins?

The function of RAR1 in race-specific resistance is conserved in plant species, including Arabidopsis, tobacco, and barley, and its genetic requirement is limited to subsets of both CC-NB-LRR and TIR-NB-LRR–type R proteins (Shirasu and Schulze-Lefert, 2003). By crossing epitope-tagged Mla6 or Mla1 transgenic lines into the rar1 mutant background, we have demonstrated that the steady state levels of both isoforms are equally dependent on RAR1, although Mla1 function is genetically independent of Rar1 (Jørgensen, 1996). This suggests that the involvement of RAR1 in R gene–triggered resistance is broader than indicated by previous genetic studies. Consistent with this, Rx resistance in N. benthamiana against PVX virus was found to be genetically independent of Rar1, but Rx abundance was dramatically reduced in Rar1 silenced plants (Figure 5A). In Arabidopsis, the abundance of RPM1, which recognizes Pseudomonas syringae AvrRpm1, is also reduced in nonchallenged rar1 mutant plants. However, similar to MLA6, this reduced R protein accumulation is accompanied by an impaired immune response (Tornero et al., 2002b). Collectively, a reduction of NB-LRR protein steady state levels in nonchallenged plants appears to be a common feature of plants lacking RAR1 irrespective of whether RAR1 is genetically essential for the resistance response.

To further examine the differential genetic requirement and general biochemical involvement of RAR1 in NB-LRR–mediated resistance, we used MLA chimeras that were generated by domain swap experiments between MLA1 and MLA6. The two chimeras tested, each recognizing AvrMla6, showed in resistance assays either an enhanced requirement for or uncoupling from Rar1 in comparison to wild-type Mla6 (Shen et al., 2003). The abundance of these two chimeras differed and inversely correlated with their genetic dependence for Rar1. Furthermore, a reduction of Mla1 gene dosage from two to one resulted in compromised Mla1 resistance in rar1 mutant plants (i.e., Mla1 function became haploinsufficient in the absence of RAR1) (Figure 4F). Taken together, these findings implicate the existence of a threshold for R protein–derived signal to trigger effective resistance and a decisive function of RAR1 in this process. The new data suggest an activity for RAR1 that is distinct from its previously inferred role in the signaling of resistance responses (Jørgensen, 1996; Shirasu et al., 1999; Halterman and Wise, 2004; Liu et al., 2004).

If RAR1 exerts a cochaperone-like activity (Shirasu and Schulze-Lefert, 2003), its role in elevating NB-LRR protein abundance might be in folding of R proteins into a form competent for effector recognition and activation of the resistance response. If so, plant cells might contain both recognition inactive and recognition competent (preactivated) NB-LRR protein forms (Schulze-Lefert, 2004). However, RAR1 is unlikely to stabilize R proteins through direct physical associations because we failed to identify a direct interaction with MLA baits in our yeast two-hybrid studies and because coexpression of MLA and RAR1 in yeast was insufficient to alter R protein abundance (data not shown). This implies the need for additional plant component(s) that act together with RAR1 to elevate MLA steady state levels.

Direct and Isoform-Dependent Interactions between the MLA LRR-CT Region and SGT1

RAR1 physically interacts with SGT1 through the C-terminal CHORD-II of RAR1 and the central CS domain of SGT1 (Azevedo et al., 2002). Both proteins also interact with cytosolic HSP90 (Hubert et al., 2003; Takahashi et al., 2003). Our yeast two-hybrid studies showed that barley or Arabidopsis SGT1 interacts with the LRR-CT of MLA1 and chimera 11166 but not with MLA6 or chimera 11666, indicating MLA isoform-dependent associations. These interactions cannot involve heterologous RAR1 because yeast lacks a RAR1 homolog. Similarly, because cytosolic HSP90 was found to interact with all four tested MLA LRR-CT variants, highly sequence-related heterologous yeast HSP90 might participate in but cannot serve as determinant of MLA isoform-specific assemblies with SGT1. We conclude that subtle sequence-specific differences in the LRR are critical for associations with SGT1. This is consistent with the observation that a thermosensitive single amino acid substitution in the LRR of yeast adenylate cyclase, Cyr1p, abolished in vivo interactions with yeast SGT1 (Dubacq et al., 2002).

Halterman and Wise (2004) identified a single amino acid substitution in the MLA6 LRR (G721D) that alleviated the resistance from Rar1 dependence. Based on our data, we predict that this mutant form of MLA6 accumulates more than the wild-type protein. However, uncoupling of RAR1 dependence in MLA6 (G721D) may not merely reflect altered SGT1 binding because we failed to detect interactions between SGT1 and the LRR-CTs of wild-type MLA6 and MLA6 (G721D) in yeast two-hybrid experiments (data not shown).

MLA variants interacting with SGT1 (MLA1 and chimera 11166) seemingly do not require SGT1 for function, whereas resistance triggered by MLA6 or chimera 11666 was significantly compromised upon transient Sgt1 single cell silencing (Azevedo et al., 2002; Shen et al., 2003). However, it is conceivable that, in analogy to RAR1, all MLA variants engage SGT1. Because it is likely that the depletion of SGT1 was incomplete in cells undergoing silencing, reduced SGT1 activity might still be sufficient for MLA isoforms that interact strongly with SGT1, but not for others with weaker interaction. A differential Sgt1 dependence might therefore reflect variation in binding between the LRR-CT and SGT1.

We note that the associations involving both HSP90 and SGT1 with the C-terminal half of MLA proteins require the absence of the CC-NB, which is strongly indicative of intramolecular R protein domain interactions. Such interactions have been shown for Rx in planta, involving the LRR and NB, as well as the CC and NB-LRR. These intramolecular interactions are thought to become sequentially disrupted upon Rx activation (Moffett et al., 2002). Thus, isoform-specific in planta interactions between MLA and SGT1 are expected to be transient or absent before activation of the R proteins. Consistent with this, we failed to coimmunoprecipitate SGT1 in extracts derived from nonchallenged MLA1-HA or MLa6-HA expressing barley lines with anti-HA antibodies (data not shown), but refined future experiments will need to clarify a role for SGT1-MLA interactions in the formation of recognition-competent R forms and/or in the signaling of resistance responses after pathogen recognition.

In yeast, SGT1 is required for proper functioning of multiple nuclear and cytoplasmic targets, including the nuclear kinetochore complex, the SCF ubiquitination complex, and the LRR containing adenylate cyclase Cyr1p (Kitagawa et al., 1999; Dubacq et al., 2002; Schadick et al., 2002). In Arabidopsis, absence of SGT1b enhances defects in auxin signaling in plants containing a partially inactive TIR1 F-box protein, indicating an SGT1 function unrelated to disease resistance (Gray et al., 2003). Activation of cell death by membrane-resident Cf-4 or Cf-9 R proteins, containing extracellular LRRs, using cognate Cladosporium fulvum avirulence peptides in N. benthamiana requires intracellular SGT1, thereby revealing a SGT1 subfunction in disease resistance that must occur independently from a direct interaction with LRRs of the R protein (Peart et al., 2002). The previously reported impaired resistance upon silencing of SGT1 without detectable changes of Rx abundance in healthy N. benthamiana plants (Lu et al., 2003) and the dramatic reduction in Rx steady state levels observed in this study upon silencing of RAR1 without accompanying suppression of the resistance (Figure 5A), favors distinct roles for RAR1 and SGT1 in the formation of recognition-competent Rx forms and in resistance signaling after pathogen effector recognition.

Why do steady state levels of highly sequence-related MLA1 and MLA6 proteins differ fourfold in the presence and absence of RAR1 in nonchallenged plants? Retained isoform-specific abundance in plants coexpressing both R proteins suggests that this is directed by intrinsic R protein properties rather than by the existence of a rate-limiting common factor(s) that might be a constituent(s) of preactivated MLA complexes. The more rapid turnover of MLA6 compared with MLA1 upon exposure of the plants to elevated temperature is indicative of inherent differences in folding processes and/or stability of protein–protein interactions. Likewise, the differential steady state levels of MLA1 and chimera 11166 compared with MLA6 and 11666 could reflect a cochaperone-like activity of SGT1 in concert with HSP90. In this scenario, one of several SGT1 activities would include the formation of recognition-competent MLA.

Decay of both MLA1 and MLA6 (but not of RAR1, SGT1, and HSP90) upon exposure to elevated temperature indicates that rapid turnover of these R proteins presumably is the default. It is conceivable that RAR1, SGT1, and HSP90 rescue R proteins from default decay and enable regulated abundance sufficient for effective resistance but below levels that could result in autoactivation of resistance responses as observed upon overexpression of Rx or Arabidopsis RPS2 (Tao et al., 2000; Bendahmane et al., 2002).

Plants have relatively few NB-LRR genes available to recognize a large number of rapidly evolving pathogen effector molecules (Meyers et al., 2003). Consequently, diversifying selection acts on some of these plant R genes to persist in the arms race with the pathogen (Dodds et al., 2004). However, diversified recognition capability might be possible only at the cost of structural instability. Interestingly, HSP90 proteins have been shown in animals and plants to buffer phenotypic variation (Rutherford, 2003). Thus, it is conceivable that HSP90 together with RAR1 and SGT1 might buffer such structural instability and widen the conformational limits of NB-LRR proteins to allow more functional variants and, therefore, more recognition specificities.

Plant Material, Fungal Isolates, and Plant Transformation

Transgenic barley lines were generated by Agrobacterium tumefaciens–mediated transformation of immature embryos derived from Hordeum vulgare ssp vulgare (barley) cultivar Golden Promise essentially as described (Wang et al., 2001; S. Schulze and H. Steinbiß, unpublished data). Transgenic plants were analyzed by DNA gel blots: DNA was extracted from young leaf material with the Nucleon PhytoPure Genomic DNA extraction kit (Amersham Biosciences, Piscataway, NJ), 20 μg of DNA was digested with HindIII (New England Biolabs, Beverly, MA), resolved in 0.7% TEA agarose gels, and blotted on nylon membranes (Roche, Penzberg, Germany) using SSC as described (Maniatis et al., 1982). Membranes were hybridized with probes matching the 5′ part of Mla1 intron 3 (primers 5′-CATTATATTTCCATGCATGCC-3′ and 5′-ACACATCAGAAAGCTGAGGG-3′) and Hpt (primers 5′-CCTCGGACGAGTGCTGG-3′ and 5′-ATTTCGTGTGTCTATGATGATG-3′) using the DIG system (Roche) according to the manufacturer's protocol. The three-component transient single-cell transformation assay to test epitope-tagged Mla variants was performed as described (Zhou et al., 2001). The vector V26-UMUG was used to express the Mlo and β-glucuronidase genes, both driven by the maize (Zea mays) ubiquitin promoter, in barley cultivar Ingrid mlo3 or Ingrid mlo5 seedling. Mla1 function was tested with Blumeria graminis f. sp hordei isolate K1 (AvrMla1, AvrMla3, AvrMla7, AvrMla13, and AvrMla22) and Mla6 function with isolate A6 (AvrMla3, AvrMla6, AvrMla7, AvrMla9, AvrMla10, AvrMla12, and AvrMla13). The resistance phenotype of the different genotypes was assayed by infecting 7-d-old seedlings with low density of Bgh spores of the compatible or incompatible fungal isolate. The resistance response was scored 7 d postinoculation.

Plasmid Construction

A pBluescript II KS− derivative with AscI and NotI sites flanking the polycloning site (pBS+AN) was used to subclone ∼8-kb fragments containing Mla1 (pBS-Mla1; with SacII-XhoI derived from cosmid p6-49-2; Zhou et al., 2001) or Mla6 (pBS-Mla6; with AvrII-PciI derived from cosmid 9589-5a; Halterman et al., 2001). N-terminal epitope tags were introduced by PCR with primers 5′-GATTGAAGCGACCCTCAC-3′ (Mla1 exon 2) or 5′-ATAAGTGCTTTTGAGTACTTGC-3′ (Mla6 exon 1) and 5′-TACCGACCGGTGACAATATCGGCTAAATCTTCCTCACTTATTAATTTTTGTTCCATGAGAGCAGGAGGAC-3′ (AgeI site underlined, start codon bold) to introduce the c-myc epitope or 5′-TACCGACCGGTGACAATATCGGCAGCATAATCTGGAACATCGTATGGATACATGAGAGCAGGAGGAC-3′ for the HA epitope. The HindIII-AgeI (Mla1) or AflII-AgeI (Mla6) fragment, respectively, of these PCR products was ligated into pBS-Mla1 or pBS-Mla6.

To generate the C-terminal epitope-tagged variants, overlapping PCR products (PCR a and b) were extended with a final PCR reaction, cut SbfI-EcoRI or BspEI-XhoI and ligated into pBS-Mla1 or pBS-Mla6, respectively. Primers 5′ and 3′ of the stop codon of Mla1 (PCR a forward, 5′-GTAAGAGTTTGGTTCAGCC-3′, and PCR b reverse, 5′-TACAATCAACACCCCTAGG-3′) or Mla6 (PCR a forward, 5′-ATAAGTGCTTTTGAGTACTTGC-3′, and PCR b reverse, 5′-CTTTTCGGTAAAACAACACTCC-3′) were used together with internal primers to generate two PCR products that cover the c-myc epitope (Mla1 PCR a, 5′-TAAATCTTCCTCACTTATTAATTTTTGTTCAGCGTTCTCCTCCTCGTCCTC-3′; Mla6 PCR a, 5′-TAAATCTTCCTCACTTATTAATTTTTGTTCAGCGTTCTCCTCCTCGCCCTC-3′; PCR b, 5′-GAACAAAAATTAATAAGTGAGGAAGATTTATGATTTCTGATCCAGAGCG-3′; epitope underlined, stop codon in bold), the HA epitope (Mla1 PCR a, 5′-AGCATAATCTGGAACATCGTATGGATAAGCGTTCTCCTCCTCGTCCTC-3′; Mla6 PCR a, 5′-AGCATAATCTGGAACATCGTATGGATAAGCGTTCTCCTCCTCGCCCTC-3′; PCR b, 5′-GCTTATCCATACGATGTTCCAGATTATGCTTGATTTCTGATCCAGAGCG-3′), or a NcoI-BamHI-HindIII adapter to insert other tags (Mla1 PCR a, 5′-ATAAGCTTTTGGATCCGCCGCCCATGGCGTTCTCCTCCTCGTCCTC-3′; Mla6 PCR a, 5′-ATAAGCTTTTGGATCCGCCGCCCATGGCGTTCTCCTCCTCGCCCTC-3′; PCR b, 5′-GAGGAGGAGAACGCCATGGGCGGCGGATCCAAAAGCTTATGATTTCTGATCCAGAGCGACTC-3′; restriction sites underlined). The NcoI-BamHI-HindIII adapter was used to introduce the myc-HA tag (5′-CATGGGCGAGCAGAAGCTCATCTCCGAGGAGGACCTCGGCTACCCGTACGACGTGCCGGACTACGCCT-3′ and 5′-GATCAGGCGTAGTCCGGCACGTCGTACGGGTAGCCGAGGTCCTCCTCGGAGATGAGCTTCTGCTCGCC-3′ were hybridized and cloned into NcoI-BamHI; epitope sequences underlined), the triple-HA tag (5′-CCATGGCCTACCCGTACGACGTGCCGGACTACGCCGGTGGTTACCCGTATGATGTACCCGATTATGCG-3′ and 5′-GGATCCTTAGGCGTAGTCCGGCACGTCGTACGGGTAACCACCCGCATAATCGGGTACATCAT-3′ were hybridized, extended with polymerase, cut, and ligated with NcoI-BamHI), GFP (NcoI-BamHI fragment from pCAT-GFP, a gift from J. Müller, MPI Cologne, Germany), GFP-HA (PCR with primers 5′-GATAGCCATGGGTAAAGGAGAAG-3′ and 5′-CGCGCGGATCCTTAGGCGTAGTCCGGCACGTCGTACGGGTAGGCTTTGTATAGTTCATCCATGCCA-3′ on pCAT-GFP, inserted as NcoI-BamHI fragment), the TAP-tag (a NcoI-HindIII fragment was subcloned from plasmid pBS1539 provided by EMBL, Heidelberg, Germany; Rigaut et al., 1999), GFP-TAP-tag (overlapping PCR products a with primers 5′-GATAGCCATGGGTAAAGGAGAAG-3′ and 5′-CTTTTTCCATCTTCTCTTTTCCATTTTGTATAGTTCATCCATGCC-3′ on plasmid pCAT-GFP and b with primers 5′-GGCATGGATGAACTATACAAAATGGAAAAGAGAAGATGGAAAAAG-3′ and 5′-GGTCCGGATCCTCAGGTTGACTTCCCCGCGGAGTTCGCGTCTAC-3′ on pBS1539 were hybridized, extended in a final PCR reaction and ligated as NcoI-BamHI fragment), and TAP-GFP-HA (overlapping PCR products a with primers 5′-ACGATCCATGGAAAAGAGAAGA-3′ and 5′-ACGATCCATGGAAAAGAGAAGA-3′ on plasmid pBS1539 and b with primers 5′-TCCGCGGGGAAGTCAACGATGGGTAAAGGAGAAGAACTTTTC-3′ and 5′-CGCGCGGATCCTTAGGCGTAGTCCGGCACGTCGTACGGGTAGGCTTTGTATAGTTCATCCATGCCA-3′ on plasmid pCAT-GFP were hybridized, extended in a final PCR reaction and ligated as NcoI-BamHI fragment). Mla chimeras 11166 and 11666 were placed under Mla6 native regulatory sequences and fused to a C-terminal HA epitope by subcloning AgeI-BspEI fragments from plasmids pUbi-11166 and pUbi-11666 (Shen et al., 2003) into the C-terminal HA epitope tag derivative of pBS-Mla6 (see above). Mla1, Mla6, their epitope-tagged derivatives, as well as the epitope-tagged chimeras were subcloned as AscI-NotI fragments into the Agrobacterium binary vector pWBVec8+A, which contains an additional unique AscI site in proximity to the unique NotI site.

Genotyping and Semiquantitative RT-PCR

Genomic DNA was extracted from leaf material (Cone, 1989) and subjected to PCR to test the presence of Mla1 or Mla6 with a C-terminal epitope (5′-GGATGGTAACCGTGGCTTC-3′ with 5′-ATAATCTGGAACATCGTATGG-3′ for the HA epitope and 5′-TTCCTCACTTATTAATTTTTGTTC-3′ for the myc epitope). A cleaved-amplified polymorphic sequence marker was used to detect the rar1-2 mutation: PCR amplification with 5′-AGGCAATCCCAAATTCGA-3′ and 5′-CCACTGCCTGCCATGGGA-3′ followed by digest with AlwNI (New England Biolabs) leads to a cleaved product for the wild-type gene that is uncleaved in rar1-2 genotypes.

For RT-PCR, total RNA was extracted using the Trizol reagent according to the provider's instructions (Invitrogen, Carlsbad, CA). Five micrograms of RNA was subjected to reverse transcription (SuperScript II; Invitrogen) with a polyT primer and PCR with MLA-HA specific primers spanning intron 4 (see above). PCR products were resolved in agarose gels after 20, 25, 30, and 35 cycles. The control PCR on actin was performed with primers 5′-TGGCACCCGAGGAGCACC-3′ and 5′-GTAACCTCTCTCGGTGAG-3′.

SDS-PAGE and Immunoblotting

Crude protein extract was produced from total primary leaf tissue of 10-d-old seedlings or from tissues as indicated. Samples were ground in liquid nitrogen, thawed in two volumes of extraction buffer (50 mM Hepes, pH 7.5, 150 mM NaCl or as indicated, 10 mM EDTA, 5 mM l-ascorbic acid, 5 mM 1,4-dithiothreitol, and 1× complete protease inhibitor cocktail from Roche) and cleared from debris by two centrifugation steps at 16,000g for 5 min. The separation into soluble and microsomal fractions included ultracentrifugation at 100,000g for 1 h and the subsequent resolubilization of the pellet in the original volume of extraction buffer using an ultrasonic bath (in ice water). Protein concentration was determined with the Bio-Rad protein assay (Hercules, CA). Proteins were separated in a 7% SDS polyacrylamide gel (12% for experiments to detect RAR1, SGT1, or ROR2) and blotted to nitrocellulose membrane (Hybond ECL; Amersham Biosciences), both using the Mini-Protean II system (Bio-Rad). Blots were incubated with primary antibodies against the HA epitope (rat monoclonal, clone 3F10, dilution 1:5000; Roche), c-myc epitope (rabbit polyclonal, 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), RAR1 (rat polyclonal, 1:2000; Takahashi et al., 2003), HSP90 (rat polyclonal, 1:10000; Takahashi et al., 2003), SGT1 (rabbit polyclonal, 1:10000; Takahashi et al., 2003), ROR2 (rabbit polyclonal, 1:250; Collins et al., 2003), and COI1 (rabbit polyclonal, 1:100; Devoto et al., 2002) followed by anti-rat/rabbit IgG (whole molecule)-peroxidase (Sigma, St. Louis, MO), and signals were detected with the ECL chemiluminescent protein gel blotting detection reagents (Amersham Biosciences). Protein gel blot signals were quantified with a CCD camera (Lumi-Imager and LumiAnalyst 3.0 software; Boehringer Mannheim/Roche).

Yeast Two-Hybrid Screening

Fusion baits were constructed using Mla1, Mla6, or chimeric full-length cDNA or partial cDNA fragments fused to the LexA DNA binding domain in the pLexA bait vector (Azevedo et al., 2002). Using the LiAc transformation method (Gietz and Woods, 1998), individual MLA bait constructs were transformed into the yeast strain EGY48 (mating type, MATα) that carries an autonomous plasmid (p8op-LacZ) in which the LacZ reporter gene is integrated. All used bait plasmids did not activate the reporter gene by themselves. A barley prey library was created using cDNA synthesized from poly(A)⁺ RNA isolated from mixed leaf tissue samples of barley cultivar Sultan5. Directional, poly(dT)₁₇-primed cDNA with EcoRI/XhoI adapters (Stratagene, La Jolla, CA) was ligated into the corresponding sites of the prey vector pB42AD (CLONTECH, Palo Alto, CA) according to the manufacturer (P. Piffanelli and P. Schulze-Lefert, unpublished data). High purity library DNA was obtained and used to transform the YM4271 strain (mating type, MATa). In total, ∼2 × 10⁶ independent yeast transformants were obtained from SD/-Trp selection plates. Glycerol stocks of this library were kept as individual aliquots and used for subsequently mating type protein–protein interaction screens as described (Kolonin et al., 2000). More details about the screen and other potential interactors recovered will be described elsewhere. The prey vectors expressing HvRAR1, HvSGT1, AtSGT1a, AtSGT1b, and HvHSP90 have been described previously (Azevedo et al., 2002; Takahashi et al., 2003).

TRV-Mediated Gene Silencing in Nicotiana benthamiana

The Rx-4HA transgenic N. benthamiana plants are described elsewhere (Lu et al., 2003). The TRV:RAR1 construct and virus-induced gene silencing initiation is as described elsewhere except, importantly, plants were sampled at 13 to16 d postinoculation rather than 21 d postinoculation (Schornack et al., 2004). Protein extracts were prepared by grinding 0.5 g of leaf tissue in 1 mL of extraction buffer (25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 10% glycerol, and 5 mM DTT). Samples were run and blotted using NuPAGE gels (Invitrogen) according to the manufacturer's guidelines. Rx levels were determined by detecting the HA epitope tag using 3F10 antibody (Roche) as previously described (Moffett et al., 2002). RAR1 levels were determined using custom antibody EP031453 (Eurogentec, Seraing, Belgium) generated using the peptide antigen GCKTGKHTTEKPVLAKS that corresponds to the CHORD I domain of AtRAR1.

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