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Regulatory Role of SGT1 in Early R Gene-Mediated Plant Defenses

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Science  15 Mar 2002:
Vol. 295, Issue 5562, pp. 2077-2080
DOI: 10.1126/science.1067747

Abstract

Animal SGT1 is a component of Skp1-Cullin–F-box protein (SCF) ubiquitin ligases that target regulatory proteins for degradation. Mutations in one (SGT1b) of two highly homologousArabidopsis SGT1 genes disable early plant defenses conferred by multiple resistance (R) genes. Loss ofSGT1b function in resistance is not compensated for bySGT1a. R genes differ in their requirements forSGT1b and a second resistance signaling gene,RAR1, that was previously implicated as an SGT1 interactor. Moreover, SGT1b and RAR1 contribute additively toRPP5-mediated pathogen recognition. These data imply both operationally distinct and cooperative functions of SGT1 and RAR1 in plant disease resistance.

In plants, a major form of resistance to disease caused by microbial pathogens is by expression of complementary gene pairs in the plant and pathogen, known respectively as resistance (R) and avirulence (avr) genes (1). Direct or indirect interaction of their products activates cellular defenses that prevent pathogen colonization of the plant. Failure to express either component results in disease susceptibility. The predominant class ofR gene in a diverse range of species encodes cytoplasmic proteins that resemble Nod proteins involved in animal innate immunity (1, 2). Plant Nod-like proteins possess a central nucleotide-binding (NB)/apoptotic adenosine triphosphatase–homologous domain, COOH-terminal leucine-rich repeats (LRRs), and NH2-terminal portions that either resemble the cytoplasmic domains of Drosophila and human Toll-like receptors [Toll–interleukin-1–resistance (TIR) domains] or contain a coiled-coil (CC) motif. Resistance conditioned by NB-LRR proteins is commonly associated with rapid, localized programmed cell death known as the hypersensitive response (HR) and an oxidative burst producing reactive oxygen intermediates (ROI) (3).

The RPP5 gene in Arabidopsis accession Landsbergerecta (Ler) encodes a TIR–NB-LRR protein conferring resistance to the downy mildew pathogen Peronospora parasitica (4). We undertook mutational screens to identify other essential genes in RPP5 resistance and have identified multiple, independent recessive mutations at four loci (5). Two of these, EDS1 and PAD4, encode lipase-like proteins that are engaged by TIR–NB-LRR type proteins but are dispensible in resistance conditioned by CC–NB-LRR proteins (6–9). The third gene is the singleArabidopsis ortholog of barley RAR1(10), a necessary component of R gene–mediated resistance to the powdery mildew fungus (11).RAR1 encodes a 25-kD cytoplasmic protein with two zinc-binding CHORDs (cysteine- and histidine-rich domains) that are conserved in sequence and tandem organization among all eukaryotic phyla examined (12). Metazoan CHORD proteins possess a motif (the CS domain) that is exclusively shared with SGT1 (12), an essential component of cell cycle progression via SKP1-mediated activation of the kinetochore complex CBF3 in yeast (13). Yeast SGT1 also associates with the SCF ubiquitin ligase complex by interaction with SKP1 and is required for SCF-mediated protein ubiquitylation in vitro (13).

We identified the fourth locus essential for Arabidopsis RPP5 resistance as one (SGT1b) of two SGT1homologs (14). Although the predictedArabidopsis SGT1a and SGT1b proteins are highly sequence related, only mutations in SGT1b were isolated in screens for components of RPP5 resistance (Fig. 1, A and B). The conserved intron/exon structures of SGT1a and SGT1b(14), coupled with higher sequence relatedness between them than with other plant SGT1 homologs (15), suggest that they have arisen by recent gene duplication (16). A combination of RNA gel blot (Fig. 1C) and RNA quantification using Taqman chemistry (5) revealed defects in SGT1b but not SGT1atranscripts in the sgt1b mutants. Thus,SGT1b is a necessary and nonredundant component ofRPP5-mediated resistance.

Figure 1

Sequence analysis of Arabidopsis SGT1a and SGT1b. (A) Plant SGT1 has been assigned several domains (12, 15): TPR, tetratricopeptide repeats; CS, CHORD protein and SGT1–specific; SGS, SGT1-specific motif; VR1 and VR2, variable domains. Three independent mutations were identified in Arabidopsis SGT1b. Insgt1b-1, a nucleotide substitution changes Trp242 → stop, causing a predicted protein truncation.sgt1b-2 is mutated within the intron 5 consensus splice acceptor site (G1317 → A). sgt1b-3 has a single-nucleotide mutation within the intron 1 splice donor site (G161 → A). (B) Sequence alignment between Arabidopsis SGT1a and SGT1b shows 78.3% amino acid identity. SGT1a and SGT1b are, respectively, 28.8% and 27.3% identical to yeast SGT1. Black and shaded boxes indicate identical and >50% conserved residues, respectively. A black line below SGT1b amino acids 140 to 159 shows the peptide sequence used to derive SGT1b-specific antisera. The Trp242 mutation in sgt1b-1 is marked with an asterisk. Single-letter abbreviations for amino acid residues: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. (C) RNA gel blot analysis of SGT1aand SGT1b transcripts in mutant and wild-type plants. Total RNA from wild-type (Ler) and sgt1bmutant lines was probed with SGT1a- orSGT1b-specific radiolabeled cDNAs.

We examined the relative levels of SGT1a and SGT1b proteins in soluble extracts from wild-type and sgt1b mutant plants (5). Probing protein blots with antibodies raised against the conserved SGS domain of SGT1 (15) showed that SGT1b was undetectable in all three sgt1b lines (Fig. 2A). SGT1a was not depleted in thesgt1b mutants, indicating unaltered stability of SGT1a in the absence of SGT1b (Fig. 2A). SGT1b was not detectable insgt1b-1, sgt1b-2, and sgt1b-3 extracts probed with an SGT1b-specific antibody; hence, all of them may be null mutations (Fig. 2B). This conclusion is consistent with the observation that each sgt1b allele exhibited an equivalent loss ofRPP5 resistance. We reasoned that the sgt1bphenotype is likely to be due to defects specifically inSGT1b.

Figure 2

Analysis of SGT1a and SGT1b protein expression. Immunoblots of total protein extracts from wild-type Ler, sgt1bmutants, partial (rar1-14, rar1-15) and null (rar1-11) rar1 (10), eds1-2(9), and pad4-2 (8) mutant lines were probed with antibody to SGS (a-SGS) (A) that detects SGT1a and SGT1b (15) or with an antibody specific for SGT1b (a-SGT1b) (B) (5). (C) Blots of total protein extracted from Ler (RPP5) or Ler (rpp5) (4) plants after inoculation with P. parasiticaisolate Noco2 were probed with antibody to SGT1b. (D) Expression of the defense marker PR1 in Noco2-inoculated or water-treated Ler (RPP5) plants was measured usingArabidopsis antibody to PR1 (24). Extract fromcpr6, which constitutively expresses PR genes (25), was used as a positive control.T 0 and T 48 indicate time elapsed since treatment. In all panels, Rubisco (ribulose-1,5-bisphosphate carboxylase-oxygenase) was used as a protein loading control.

Levels of SGT1b were unaffected by mutations in each of theRPP5 resistance signaling components RAR1,EDS1, and PAD4 (Fig. 2B). SGT1b expression also did not change after inoculation of wild-type leaves with pathogen isolate P. parasitica Noco2 that is recognized byRPP5 (Fig. 2C). Similar results were obtained in tissues ofrpp5 mutant plants that fail to recognize Noco2 and therefore allow pathogen colonization (Fig. 2C). PR1, a marker of downstream plant defenses, was induced in inoculated wild-type (RPP5) but not in control samples (Fig. 2D), indicating that resistance was appropriately elicited in these experiments. Therefore, pathogen infection does not alter SGT1b expression substantially, although we cannot rule out that changes in SGT1b abundance occur transiently or in a restricted number of cells at pathogen penetration sites.

We compared the defects of sgt1b-1 with other mutant lines that compromise RPP5 by measuring the extent of pathogen asexual sporulation and plant cell necrosis on infected leaves. Loss ofRPP5 resistance in sgt1b was equivalent to that in a rar1 null mutant but was not as strong as in nullrpp5 or eds1 plants that exhibited, respectively, susceptibility and supersusceptibility (Fig. 3A) (4, 9). A doublesgt1b/rar1 mutant exhibited additive disease susceptibility equivalent to rpp5 (Fig. 3A), indicating that wild-typeSGT1b and RAR1 operate genetically independently of each other in RPP5 resistance. Examination of inoculated leaves under ultraviolet light revealed occasional plant cell death–associated autofluorescence in sgt1b andrar1 but not in rpp5, eds1, orsgt1b/rar1 (Fig. 3B). By microscopic analysis, we estimated that the HR was disabled in ∼95% of pathogen inoculation sites ofsgt1b (Fig. 3C) and rar1 (10), contrasting with a complete suppression of HR in rpp5 (Fig. 3C). Substantially delayed plant cell death in sgt1b was evident later in infection by appearance of necrotic plant cells trailing the pathogen (Fig. 3C). Whole-cell ROI accumulation was undetectable at most pathogen inoculation sites of sgt1b andrar1 but was occasionally observed in cells at later stages of colonization (17). These data reveal cooperation between SGT1 and RAR1 in the regulation of RPP5-triggered cell death and the oxidative burst.

Figure 3

Pathogen colonization of sgt1band rar1 mutants. (A) Asexual sporulation levels of P. parasitica isolate Noco2 7 days after inoculation of wild-type (Ler) and different RPP5 signaling mutants (5). Sporulation on sgt1b-1 andrar1-10 was significantly different (Student'st test, *P = 0.05) from that measured on sgt1b-1/rar1-10. (B) Noco2-infected leaves viewed under ultraviolet light exhibiting cell death–associated fluorescence in rar1-10 and sgt1b-1 5 days after inoculation. (C) Leaves of Ler (RPP5),sgt1b-1, and rpp5 were stained with lactophenol trypan blue at 3 and 6 days post-inoculation (dpi) with Noco2 to reveal necrotic plant cells and pathogen structures (26). HR, hypersensitive response; M, mycelium; TN, trailing plant cell necrosis; S, pathogen sporangiophore. (D) Growth of P. syringae pv. tomato strain DC3000 expressingavrRpm1, avrRps4, or containing an empty vector (DC3000) was measured in different plant lines (5): ▪,rar1-10; ▵, eds1-2; ×, sgt1b-1; ⧫, Ler. Each data point represents the average of three replicates.

In Arabidopsis, RAR1 is essential for resistance conferred by multiple R genes recognizing distinct avirulentP. parasitica or Pseudomonas syringae pv.tomato isolates (10). We found that theseR genes have either common or separate genetic requirements for RAR1 and SGT1b (Fig. 3D and Table 1). These findings provide evidence that SGT1b and RAR1 have overlapping but nonidentical roles in resistance triggered by different R genes. Usage of RAR1and/or SGT1b was not restricted to a particular NB-LRR structural type (Table 1), whereas EDS1 andPAD4 are specifically required by TIR–NB-LRR proteins (6, 9).

Table 1

Different Arabidopsis R gene requirements for SGT1b and RAR1. Disease resistance (R), susceptibility (S), or partial susceptibility [(S)] was scored on the basis of asexual sporulation levels of P. parasiticaisolates or bacterial growth of P. syringae strains (5). All R genes tested were expressed in the Ler background except RPP2 and RPP4 in accession Columbia and RPP1A in Wassileskija that were individually combined with sgt1b-1 or rar1-10 in segregating F2 populations (5, 10). TIR and CC domains of cloned NB-LRR genes are indicated (5).

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In yeast, the cellular roles of SGT1 are closely linked to its association with SKP1, an integral ubiquitin ligase protein (13). A mouse homolog of SGT1 was also identified by mass spectrometry as an SCF interactor (18). The sequence relatedness of Arabidopsis SGT1a and SGT1b (Fig. 1B), coupled with the ability of both genes to complement two yeast temperature-sensitive sgt1 mutant alleles (15), suggests that fundamental cellular function(s) of yeast SGT1in SCF-mediated protein ubiquitylation (13, 19, 20) are retained by both Arabidopsis SGT1 homologs. We present evidence that SGT1b has evolved a distinct capability in certain R gene–specified responses that is not compensated for by SGT1a. Nonredundant functions ofArabidopsis SGT1a and SGT1b in plant defense may reflect preferential interactions with different subsets of SCF complexes. Representation of at least 19 SKP1 orthologs and >300 F-box–containing proteins in the Arabidopsis genome creates the potential for considerable flexibility in SCF composition and regulatory function (16, 21–23).

Our data show that RAR1 and SGT1b each contribute quantitatively to RPP5-dependent resistance and are thus operationally distinct. It is noteworthy that Azevedo et al. (15) show conserved molecular association between plant SGT1 and RAR1, including Arabidopsis SGT1a and SGT1b. In barley extracts, SGT1 exists in two pools, one containing RAR1, the other engaging SCF components. The combined genetic and molecular data imply that SGT1-RAR1 and SGT1-SCF complexes have at least partially distinct roles in disease resistance. In one scenario, mutations in RAR1but not SGT1a or SGT1b might disable SGT1-RAR1 function, whereas mutations in SGT1b might compromise a subset of SCF complexes that are preferentially used inR gene–triggered responses. Such a model would account for the exclusive genetic requirements of certain R genes for either RAR1 or SGT1b, directing signals through one or the other mechanism to trigger plant defense.

  • * To whom correspondence should be addressed. E-mail: parker{at}mpiz-koeln.mpg.de

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