Perturbation of Arabidopsis amino acid metabolism causes incompatibility with the adapted biotrophic pathogen Hyaloperonospora arabidopsidis.

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.

Repression of the auxin response pathway increases Arabidopsis susceptibility to necrotrophic fungi.

In plants, resistance to necrotrophic pathogens depends on the interplay between different hormone systems, such as those regulated by salicylic acid (SA), jasmonic acid (JA), ethylene, and abscisic acid. Repression of auxin signaling by the SA pathway was recently shown to contribute to antibacterial resistance. Here, we demonstrate that Arabidopsis auxin signaling mutants axr1, axr2, and axr6 that have defects in the auxin-stimulated SCF (Skp1-Cullin-F-box) ubiquitination pathway exhibit increased susceptibility to the necrotrophic fungi Plectosphaerella cucumerina and Botrytis cinerea. Also, stabilization of the auxin transcriptional repressor AXR3 that is normally targeted for removal by the SCF-ubiquitin/proteasome machinery occurs upon P. cucumerina infection. Pharmacological inhibition of auxin transport or proteasome function each compromise necrotroph resistance of wild-type plants to a similar extent as in non-treated auxin response mutants. These results suggest that auxin signaling is important for resistance to the necrotrophic fungi P. cucumerina and B. cinerea. SGT1b (one of two Arabidopsis SGT1 genes encoding HSP90/HSC70 co-chaperones) promotes the functions of SCF E3-ubiquitin ligase complexes in auxin and JA responses and resistance conditioned by certain Resistance (R) genes to biotrophic pathogens. We find that sgt1b mutants are as resistant to P. cucumerina as wild-type plants. Conversely, auxin/SCF signaling mutants are uncompromised in RPP4-triggered resistance to the obligate biotrophic oomycete, Hyaloperonospora parasitica. Thus, the predominant action of SGT1b in R gene-conditioned resistance to oomycetes appears to be at a site other than assisting SCF E3-ubiquitin ligases. However, genetic additivity of sgt1b axr1 double mutants in susceptibility to H. parasitica suggests that SCF-mediated ubiquitination contributes to limiting biotrophic pathogen colonization once plant-pathogen compatibility is established.

The CLAVATA1-related BAM1, BAM2 and BAM3 receptor kinase-like proteins are required for meristem function in Arabidopsis.

Organ formation at shoot and flower meristems in plants requires the maintenance of a population of centrally located stem cells and the differentiation of peripherally located daughter cells. The CLAVATA (CLV) gene products in Arabidopsis, including the CLV1 receptor-kinase, regulate this process by promoting the differentiation of stem cells on the meristem flanks. Here, we have analyzed the developmental roles of the CLV1-related BAM1 (derived from barely any meristem 1), BAM2 and BAM3 receptor-like kinases. Loss-of-function alleles of these receptors lead to phenotypes consistent with the loss of stem cells at the shoot and flower meristem, suggesting that their developmental role is opposite to that of CLV1. These closely related receptors are further distinguished from CLV1, whose expression and function is highly specific, by having broad expression patterns and multiple developmental roles. These include a requirement for BAM1, BAM2 and BAM3 in the development of high-ordered vascular strands within the leaf and a correlated control of leaf shape, size and symmetry. In addition, BAM1, BAM2 and BAM3 are required for male gametophyte development, as well as ovule specification and function. Significantly, the differing roles of CLV1 and BAM receptors in meristem and organ development are largely driven by differences in expression patterns.

The atypical resistance gene, RPW8, recruits components of basal defence for powdery mildew resistance in Arabidopsis.

Genetic studies have identified a number of components of signal transduction pathways leading to plant disease resistance and the accompanying hypersensitive response (HR) following detection of pathogens by plant resistance (R) genes. In Arabidopsis, the majority of R proteins so far characterized belong to a plant superfamily that have a central nucleotide-binding site and C-terminal leucine-rich-repeats (NB-LRRs). Another much less prevalent class comprises RPW8.1 and RPW8.2, two related proteins that possess a putative N-terminal transmembrane domain and a coiled-coil motif, and confer broad-spectrum resistance to powdery mildew. Here we investigated whether RPW8.1 and RPW8.2 engage known pathway(s) for defence signalling. We show that RPW8.1 and RPW8.2 recruit, in addition to salicylic acid and EDS1, the other NB-LRR gene-signalling components PAD4, EDS5, NPR1 and SGT1b for activation of powdery mildew resistance and HR. In contrast, NDR1, RAR1 and PBS3 that are required for function of certain NB-LRR R genes, and COI1 and EIN2 that operate, respectively, in the jasmonic acid and ethylene signalling pathways, do not contribute to RPW8.1 and RPW8.2-mediated resistance. We further demonstrate that EDR1, a gene encoding a conserved MAPKK kinase, exerts negative regulation on HR cell death and powdery mildew resistance by limiting the transcriptional amplification of RPW8.1 and RPW8.2. Our results suggest that RPW8.1 and RPW8.2 stimulate a conserved basal defence pathway that is negatively regulated by EDR1.

Role of SGT1 in the regulation of plant R gene signalling.

Recent important discoveries in several laboratories have identified SGT1 as an essential component of R gene-mediated disease resistance in plants. The precise molecular function of SGT1 remains unknown, although sequence analysis and structural predictions reveal that SGT1 has features of co-chaperones that associate with HSP90 in animals. This review will describe the role of SGT1 in R gene-mediated plant defence and discuss how SGT1 may regulate this process.

Role of ubiquitination in the regulation of plant defence against pathogens.

Ubiquitination is emerging as a common regulatory mechanism that controls a range of cellular processes in plants. Recent exciting discoveries from several laboratories suggest that ubiquitination may also play an important role in plant disease resistance. Several putative ubiquitin ligases have been identified as defence regulators. In addition, a combination of genetic screens and gene-silencing technologies has identified subunits and proposed regulators of SCF ubiquitin ligases as essential components of resistance (R)-gene-mediated resistance. Although no ubiquitin ligase targets that are associated with disease resistance have yet been identified in plants, there is evidence that this well-known protein-modification system may regulate plant defences against pathogens.

A resource of mapped dissociation launch pads for targeted insertional mutagenesis in the Arabidopsis genome.

We describe a new resource for targeted insertional mutagenesis in Arabidopsis using a maize (Zea mays) Activator/Dissociation (Ds) two-element system. The two components of the system, T-DNA vectors carrying a Ds launch pad and a stable Activator transposase source, were designed to simplify selection of transposition events and maximize their usefulness. Because Ds elements preferentially transpose to nearby genomic sites, they can be used in targeted mutagenesis of linked genes. To efficiently target all genes throughout the genome, we generated a large population of transgenic Arabidopsis plants containing the Ds launch pad construct, identified lines containing single Ds launch pad inserts, and mapped the positions of Ds launch pads in 89 lines. The integration sites of the Ds launch pads were relatively evenly distributed on all five chromosomes, except for a region of chromosomes 2 and 4 and the centromeric regions. This resource therefore provides access to the majority of the Arabidopsis genome for targeted tagging.

Arabidopsis SGT1b is required for SCF(TIR1)-mediated auxin response.

The SCF(TIR1) complex is a central regulator of the auxin response pathway in Arabidopsis. This complex functions as a ubiquitin protein ligase that targets members of the auxin/indoleacetic acid (Aux/IAA) family of transcriptional regulators for ubiquitin-mediated degradation in response to auxin. In an attempt to identify additional factors required for SCF(TIR1) activity, we conducted a genetic screen to isolate enhancers of the auxin response defect conferred by the tir1-1 mutation. Here, we report the identification and characterization of the eta3 mutant. The eta3 mutation interacts synergistically with tir1-1 to strongly enhance all aspects of the tir1 mutant phenotype, including auxin inhibition of root growth, lateral root development, hypocotyl elongation at high temperature, and apical dominance. We isolated the ETA3 gene using a map-based cloning strategy and determined that ETA3 encodes SGT1b. SGT1b was identified recently as a factor involved in plant disease resistance signaling, and SGT1 from barley and tobacco extracts was shown to interact with SCF ubiquitin ligases. We conclude that ETA3/SGT1b is required for the SCF(TIR1)-mediated degradation of Aux/IAA proteins.

Arabidopsis RAR1 exerts rate-limiting control of R gene-mediated defenses against multiple pathogens.

We have identified the Arabidopsis ortholog of barley RAR1 as a component of resistance specified by multiple nucleotide binding/Leu-rich repeat resistance (R) genes recognizing different bacterial and oomycete pathogen isolates. Characterization of partially and fully defective rar1 mutations revealed that wild-type RAR1 acts as a rate-limiting regulator of early R gene-triggered defenses, determining the extent of pathogen containment, hypersensitive plant cell death, and an oxidative burst at primary infection sites. We conclude that RAR1 defense signaling function is conserved between plant species that are separated evolutionarily by 150 million years. RAR1 encodes a protein with two zinc binding (CHORD) domains that are highly conserved across eukaryotic phyla, and the single nematode CHORD-containing homolog, Chp, was found previously to be essential for embryo viability. An absence of obvious developmental defects in null Arabidopsis rar1 mutants favors the notion that, in contrast, RAR1 does not play a fundamental role in plant development.

Regulatory role of SGT1 in early R gene-mediated plant defenses.

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 homologous Arabidopsis SGT1 genes disable early plant defenses conferred by multiple resistance (R) genes. Loss of SGT1b function in resistance is not compensated for by SGT1a. R genes differ in their requirements for SGT1b and a second resistance signaling gene, RAR1, that was previously implicated as an SGT1 interactor. Moreover, SGT1b and RAR1 contribute additively to RPP5-mediated pathogen recognition. These data imply both operationally distinct and cooperative functions of SGT1 and RAR1 in plant disease resistance.