Johannes Stuttmann

Interaction between SGT1 and Cytosolic/Nuclear HSC70 Chaperones Regulates Arabidopsis Immune Responses

The conserved eukaryotic protein SGT1 (for Suppressor of G2 allele of skp1) has characteristics of an HSP90 (for heat shock protein 90 kD) cochaperone and in plants regulates hormone responses and Resistance gene–triggered immunity. We affinity-purified SGT1-interacting proteins from Arabidopsis thaliana leaf extracts and identified by mass spectrometry cytosolic heat shock cognate 70 (HSC70) chaperones as the major stable SGT1 interactors. Arabidopsis SGT1a and SGT1b proteins associate with HSC70 in vivo and distribute with HSC70 in the cytosol and nucleus. An intact C-terminal SGT1-specific (SGS) domain that is required for all known SGT1b functions in immunity and development is needed for HSC70 interaction and for the nuclear accumulation of SGT1b. Interaction assays of transiently expressed proteins or their domains in Nicotiana benthamiana point to a role of SGT1 as a HSC70 cofactor. Expression of two HSC70 isoforms is upregulated by pathogen challenge, and while loss of function of individual cytosolic HSC70 genes has no defense phenotype, HSC70-1 overexpression disables resistance to virulent and avirulent pathogens. Moreover, mutations in SGT1b lead to a similar degree of heat shock tolerance as deregulation of HSC70-1. We conclude that an HSC70-SGT1 chaperone complex is important for multiple plant environmental responses and that the evolutionarily conserved SGS domain of SGT1 is a key determinant of the HSC70–SGT1 association.

Perturbation of Arabidopsis Amino Acid Metabolism Causes Incompatibility with the Adapted Biotrophic Pathogen Hyaloperonospora arabidopsidis

This work identified two different Arabidopsis mutants that have reduced susceptibility to an infectious biotrophic pathogen due to overaccumulation of the amino acid Thr. This is detrimental for the host plant and the infecting pathogen but does not affect disease caused by some other pathogen species. Therefore, the host metabolic state can influence disease in quite a specific manner. Reliance of biotrophic pathogens on living plant tissues to propagate implies strong interdependence between host metabolism and nutrient uptake by the pathogen. However, factors determining host suitability and establishment of infection are largely unknown. We describe a loss-of-inhibition allele of ASPARTATE KINASE2 and a loss-of-function allele of DIHYDRODIPICOLINATE SYNTHASE2 identified in a screen for Arabidopsis thaliana mutants with increased resistance to the obligate biotrophic oomycete Hyaloperonospora arabidopsidis (Hpa). Through different molecular mechanisms, these mutations perturb amino acid homeostasis leading to overaccumulation of the Asp-derived amino acids Met, Thr, and Ile. Although detrimental for the plant, the mutations do not cause defense activation, and both mutants retain full susceptibility to the adapted obligate biotrophic fungus Golovinomyces orontii (Go). Chemical treatments mimicking the mutants’ metabolic state identified Thr as the amino acid suppressing Hpa but not Go colonization. We conclude that perturbations in amino acid homeostasis render the mutant plants unsuitable as an infection substrate for Hpa. This may be explained by deployment of the same amino acid biosynthetic pathways by oomycetes and plants. Our data show that the plant host metabolic state can, in specific ways, influence the ability of adapted biotrophic strains to cause disease.

Structural Basis for Signaling by Exclusive EDS1 Heteromeric Complexes with SAG101 or PAD4 in Plant Innate Immunity.

Biotrophic plant pathogens encounter a postinfection basal resistance layer controlled by the lipase-like protein enhanced disease susceptibility 1 (EDS1) and its sequence-related interaction partners, senescence-associated gene 101 (SAG101) and phytoalexin deficient 4 (PAD4). Maintainance of separate EDS1 family member clades through angiosperm evolution suggests distinct functional attributes. We report the Arabidopsis EDS1-SAG101 heterodimer crystal structure with juxtaposed N-terminal α/β hydrolase and C-terminal α-helical EP domains aligned via a large conserved interface. Mutational analysis of the EDS1-SAG101 heterodimer and a derived EDS1-PAD4 structural model shows that EDS1 signals within mutually exclusive heterocomplexes. Although there is evolutionary conservation of α/β hydrolase topology in all three proteins, a noncatalytic resistance mechanism is indicated. Instead, the respective N-terminal domains appear to facilitate binding of the essential EP domains to create novel interaction surfaces on the heterodimer. Transitions between distinct functional EDS1 heterodimers might explain the central importance and versatility of this regulatory node in plant immunity.

COP9 signalosome- and 26S proteasome-dependent regulation of SCFTIR1 accumulation in Arabidopsis.

Ubiquitination and proteasome-mediated degradation of proteins are crucial for eukaryotic physiology and development. The largest class of E3 ubiquitin ligases is made up of the cullin-RING ligases (CRLs), which themselves are positively regulated through conjugation of the ubiquitin-like peptide RUB/NEDD8 to cullins. RUB modification is antagonized by the COP9 signalosome (CSN), an evolutionarily conserved eight-subunit complex that is essential in most eukaryotes and cleaves RUB from cullins. The CSN behaves genetically as an activator of CRLs, although it abolishes CRL activity in vitro. This apparent paradox was recently reconciled in different organisms, as the CSN was shown to prevent autocatalytic degradation of several CRL substrate adaptors. We tested for such a mechanism in the model plant Arabidopsis by measuring the impact of a newly identified viable csn2 mutant on the activity and stability of SCFTIR1, a receptor to the phytohormone auxin and probably the best characterized plant CRL. Our analysis reveals that not only the F-box protein TIR1 but also relevant cullins are destabilized in csn2 and other Arabidopsis csn mutants. These results provide an explanation for the auxin resistance of csn mutants. We further observed in vivo a post-translational modification of TIR1 dependent on the proteasome inhibitor MG-132 and provide evidence for proteasome-mediated degradation of TIR1, CUL1, and ASK1 (Arabidopsis SKP1 homolog). These results are consistent with CSN-dependent protection of Arabidopsis CRLs from autocatalytic degradation, as observed in other eukaryotes, and provide evidence for antagonist roles of the CSN and 26S proteasome in modulating accumulation of the plant CRL SCFTIR1.

HpaA from Xanthomonas is a regulator of type III secretion

The Gram‐negative plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to inject effector proteins into the host cell cytoplasm. Efficient secretion of several effector proteins depends on the cytoplasmic global T3S chaperone HpaB. In this study, we show that HpaB interacts with the virulence factor HpaA, which is secreted by the T3S system and translocated into the plant cell. HpaA promotes secretion of pilus, translocon and effector proteins and therefore appears to be an important control protein of the T3S system. Protein–protein interaction studies and the analysis of HpaA deletion derivatives revealed that the C‐terminal protein region, which contains a HpaB binding site, is crucial for the contribution of HpaA to T3S. Secretion of pilus and translocon proteins is not affected when HpaA is expressed as an N‐terminal deletion derivative that lacks the secretion and translocation signal. Our data suggest that binding of HpaA to HpaB within the bacterial cell favours secretion of extracellular components of the secretion apparatus. Secretion of HpaA presumably liberates HpaB and thus promotes effector protein secretion after assembly of the T3S apparatus.

Characterization of the Nonconserved hpaB-hrpF Region in the hrp Pathogenicity Island from Xanthomonas campestris pv. vesicatoria

The interaction of the gram-negative phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria with its host plants pepper and tomato is mediated by a type III secretion (T3S) system that translocates bacterial effector proteins into the plant cell. The T3S system is encoded by the chromosomal hrp (hypersensitive response and pathogenicity) gene cluster. Here, we report on the analysis of the hpaB-hrpF region, which encodes the novel virulence factor HpaE, the effector protein XopF1, and two proteins with unknown functions, HpaD and HpaI. Promoter and transcript analyses revealed that the corresponding genes are coexpressed with the hrp genes and that hpaD, hpaI, and xopF1 form a novel operon. In vitro and in vivo assays showed that the efficient T3S and translocation of XopF1 depends on the global T3S chaperone HpaB and the putative lytic transglycosylase HpaH, which specifically contributes to the secretion of a certain set of effectors. Taken together, our data suggest that the efficient secretion of effector proteins in X. campestris pv. vesicatoria requires the contribution of several different Hpa proteins.

Nucleocytoplasmic partitioning of tobacco N receptor is modulated by SGT1

SGT1 (Suppressor of G2 allele of SKP1) is required to maintain plant disease Resistance (R) proteins with Nucleotide-Binding (NB) and Leucine-Rich Repeat (LRR) domains in an inactive but signaling-competent state. SGT1 is an integral component of a multi-protein network that includes RACK1, Rac1, RAR1, Rboh, HSP90 and HSP70, and in rice the Mitogen-Activated Protein Kinase (MAPK), OsMAPK6. Tobacco (Nicotiana tabacum) N protein, which belongs to the Toll-Interleukin Receptor (TIR)-NB-LRR class of R proteins, confers resistance to Tobacco Mosaic Virus (TMV). Following transient expression in planta, we analyzed the functional relationship between SGT1, SIPK - a tobacco MAPK6 ortholog - and N, using mass spectrometry, confocal microscopy and pathogen assays. Here, we show that tobacco SGT1 undergoes specific phosphorylation in a canonical MAPK target-motif by SIPK. Mutation of this motif to mimic SIPK phosphorylation leads to an increased proportion of cells displaying SGT1 nuclear accumulation and impairs N-mediated resistance to TMV, as does phospho-null substitution at the same residue. Forced nuclear localization of SGT1 causes N to be confined to nuclei. Our data suggest that one mode of regulating nucleocytoplasmic partitioning of R proteins is by maintaining appropriate levels of SGT1 phosphorylation catalyzed by plant MAPK.

Staying in the fold: The SGT1/chaperone machinery in maintenance and evolution of leucine-rich repeat proteins

The conserved eukaryotic protein SGT1 (Suppressor of G2 allele of skp1) participates in diverse physiological processes such as cell cycle progression in yeast, plant immunity against pathogens and plant hormone signalling. Recent genetic and biochemical studies suggest that SGT1 functions as a novel co-chaperone for cytosolic/nuclear HSP90 and HSP70 molecular chaperones in the folding and maturation of substrate proteins. Since proteins containing the leucine-rich repeat (LRR) protein-protein interaction motif are overrepresented in SGT1-dependent phenomena, we consider whether LRR-containing proteins are preferential substrates of an SGT1/HSP70/HSP90 complex. Such a chaperone organisation is reminiscent of the HOP/HSP70/HSP90 machinery which controls maturation and activation of glucocorticoid receptors in animals. Drawing on this parallel, we discuss the possible contribution of an SGT1-chaperone complex in the folding and maturation of LRR-containing proteins and its evolutionary consequences for the emergence of novel LRR interaction surfaces.

Arabidopsis thaliana DM2h (R8) within the Landsberg RPP1-like Resistance Locus Underlies Three Different Cases of EDS1-Conditioned Autoimmunity.

Plants have a large panel of nucleotide-binding/leucine rich repeat (NLR) immune receptors which monitor host interference by diverse pathogen molecules (effectors) and trigger disease resistance pathways. NLR receptor systems are necessarily under tight control to mitigate the trade-off between induced defenses and growth. Hence, mis-regulated NLRs often cause autoimmunity associated with stunting and, in severe cases, necrosis. Nucleocytoplasmic ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) is indispensable for effector-triggered and autoimmune responses governed by a family of Toll-Interleukin1-Receptor-related NLR receptors (TNLs). EDS1 operates coincidently or immediately downstream of TNL activation to transcriptionally reprogram cells for defense. We show here that low levels of nuclear-enforced EDS1 are sufficient for pathogen resistance in Arabidopsis thaliana, without causing negative effects. Plants expressing higher nuclear EDS1 amounts have the genetic, phenotypic and transcriptional hallmarks of TNL autoimmunity. In a screen for genetic suppressors of nuclear EDS1 autoimmunity, we map multiple, independent mutations to one gene, DM2h, lying within the polymorphic DANGEROUS MIX2 cluster of TNL RPP1-like genes from A. thaliana accession Landsberg erecta (Ler). The DM2 locus is a known hotspot for deleterious epistatic interactions leading to immune-related incompatibilities between A. thaliana natural accessions. We find that DM2hLer underlies two further genetic incompatibilities involving the RPP1-likeLer locus and EDS1. We conclude that the DM2hLer TNL protein and nuclear EDS1 cooperate, directly or indirectly, to drive cells into an immune response at the expense of growth. A further conclusion is that regulating the available EDS1 nuclear pool is fundamental for maintaining homeostatic control of TNL immune pathways.

Novel aspects of COP9 signalosome functions revealed through analysis of hypomorphic csn mutants

The COP9 signalosome (CSN) is a conserved eukaryotic protein complex implicated in the regulation of cullin-RING type E3 ubiquitin ligases by cleaving the small peptide RUB/Nedd8 from cullins. However, detailed analysis of CSN physiological functions in Arabidopsis has been hampered by the early seedling-lethality of csn null mutants. We and others have now identified a number of viable hypomorphic csn mutants which start to reveal novel CSN-dependent activities in adult Arabidopsis plants.1 Here, we present a detailed comparative analysis of the csn5a-1 and csn2-5 mutants as a mean to improve understanding of CSN functions in plant cells. Our observations point to CSN-independent activities of CSN5 and suggest a role of the CSN in cytoskeleton assembly/organization.