Jasmonate and phytochrome A signaling in Arabidopsis wound and shade responses are integrated through JAZ1 stability.

Jasmonate (JA) activates plant defense, promotes pollen maturation, and suppresses plant growth. An emerging theme in JA biology is its involvement in light responses; here, we examine the interdependence of the JA- and light-signaling pathways in Arabidopsis thaliana. We demonstrate that mutants deficient in JA biosynthesis and signaling are deficient in a subset of high irradiance responses in far-red (FR) light. These mutants display exaggerated shade responses to low, but not high, R/FR ratio light, suggesting a role for JA in phytochrome A (phyA) signaling. Additionally, we demonstrate that the FR light-induced expression of transcription factor genes is dependent on CORONATINE INSENSITIVE1 (COI1), a central component of JA signaling, and is suppressed by JA. phyA mutants had reduced JA-regulated growth inhibition and VSP expression and increased content of cis-(+)-12-oxophytodienoic acid, an intermediate in JA biosynthesis. Significantly, COI1-mediated degradation of JASMONATE ZIM DOMAIN1-beta-glucuronidase (JAZ1-GUS) in response to mechanical wounding and JA treatment required phyA, and ectopic expression of JAZ1-GUS resulted in exaggerated shade responses. Together, these results indicate that JA and phyA signaling are integrated through degradation of the JAZ1 protein, and both are required for plant responses to light and stress.

ZraP is a periplasmic molecular chaperone and a repressor of the zinc-responsive two-component regulator ZraSR.

The bacterial envelope is the interface with the surrounding environment and is consequently subjected to a barrage of noxious agents including a range of compounds with antimicrobial activity. The ESR (envelope stress response) pathways of enteric bacteria are critical for maintenance of the envelope against these antimicrobial agents. In the present study, we demonstrate that the periplasmic protein ZraP contributes to envelope homoeostasis and assign both chaperone and regulatory function to ZraP from Salmonella Typhimurium. The ZraP chaperone mechanism is catalytic and independent of ATP; the chaperone activity is dependent on the presence of zinc, which is shown to be responsible for the stabilization of an oligomeric ZraP complex. Furthermore, ZraP can act to repress the two-component regulatory system ZraSR, which itself is responsive to zinc concentrations. Through structural homology, ZraP is a member of the bacterial CpxP family of periplasmic proteins, which also consists of CpxP and Spy. We demonstrate environmental co-expression of the CpxP family and identify an important role for these proteins in Salmonella's defence against the cationic antimicrobial peptide polymyxin B.

Patient and clinician satisfaction and clinical outcomes of Magseed compared with wire-guided localisation for impalpable breast lesions.

BACKGROUND: Guide-wire localisation remains the most commonly used technique for localisation of impalpable breast lesions in the UK. One alternative is magnetic seed localisation. We aimed to investigate patient and clinician satisfaction in two consecutive cohorts, describe re-excision and positive margin rates, and explore reasons for positive margins and the implications for localisation techniques. METHODS: A single-institution prospective service evaluation of two cohorts of consecutive cases of wire and then Magseed localisation was carried out. Data were collected on patient and clinician satisfaction, clinico-pathological findings, and causes of involved margins. T tests were used to compare continuous variables and Chi-squared test for satisfaction outcomes. RESULTS: 168 consecutive cases used wire-guided localisation (WGL) and 128 subsequent cases used Magseeds. Patients reported less anxiety between localisation and surgery in the Magseed group, and clinicians reported greater ease of use of Magseeds. There were no differences in lesion size, surgical complexity, or re-excision rate between the groups. In a subset of patients receiving standard wide local excision (i.e., excluding mammoplasties), the impact on margin involvement was investigated. There was no significant difference in radiological under-sizing or accuracy of localisation. However, specimen weight and eccentricity of the lesion were statistically significantly lower in the Magseed group. Despite this, re-excision rates were not significantly different (p = 0.4). CONCLUSIONS: This is the first large study of satisfaction with localisation and showed clinician preference for Magseed and a reduction in patient anxiety. It also demonstrated similar positive margin rates despite smaller specimen weights in the Magseed group. Magnetic seed localisation offers an acceptable clinical alternative to guide wire localisation. The impact on local service provision should also be considered.

Chemical warfare between leafcutter ant symbionts and a co-evolved pathogen.

Acromyrmex leafcutter ants form a mutually beneficial symbiosis with the fungus Leucoagaricus gongylophorus and with Pseudonocardia bacteria. Both are vertically transmitted and actively maintained by the ants. The fungus garden is manured with freshly cut leaves and provides the sole food for the ant larvae, while Pseudonocardia cultures are reared on the ant-cuticle and make antifungal metabolites to help protect the cultivar against disease. If left unchecked, specialized parasitic Escovopsis fungi can overrun the fungus garden and lead to colony collapse. We report that Escovopsis upregulates the production of two specialized metabolites when it infects the cultivar. These compounds inhibit Pseudonocardia and one, shearinine D, also reduces worker behavioral defenses and is ultimately lethal when it accumulates in ant tissues. Our results are consistent with an active evolutionary arms race between Pseudonocardia and Escovopsis, which modifies both bacterial and behavioral defenses such that colony collapse is unavoidable once Escovopsis infections escalate.

Genome Analysis of Two Pseudonocardia Phylotypes Associated with Acromyrmex Leafcutter Ants Reveals Their Biosynthetic Potential.

The attine ants of South and Central America are ancient farmers, having evolved a symbiosis with a fungal food crop >50 million years ago. The most evolutionarily derived attines are the Atta and Acromyrmex leafcutter ants, which harvest fresh leaves to feed their fungus. Acromyrmex and many other attines vertically transmit a mutualistic strain of Pseudonocardia and use antifungal compounds made by these bacteria to protect their fungal partner against co-evolved fungal pathogens of the genus Escovopsis. Pseudonocardia mutualists associated with the attines Apterostigma dentigerum and Trachymyrmex cornetzi make novel cyclic depsipeptide compounds called gerumycins, while a mutualist strain isolated from derived Acromyrmex octospinosus makes an unusual polyene antifungal called nystatin P1. The novelty of these antimicrobials suggests there is merit in exploring secondary metabolites of Pseudonocardia on a genome-wide scale. Here, we report a genomic analysis of the Pseudonocardia phylotypes Ps1 and Ps2 that are consistently associated with Acromyrmex ants collected in Gamboa, Panama. These were previously distinguished solely on the basis of 16S rRNA gene sequencing but genome sequencing of five Ps1 and five Ps2 strains revealed that the phylotypes are distinct species and each encodes between 11 and 15 secondary metabolite biosynthetic gene clusters (BGCs). There are signature BGCs for Ps1 and Ps2 strains and some that are conserved in both. Ps1 strains all contain BGCs encoding nystatin P1-like antifungals, while the Ps2 strains encode novel nystatin-like molecules. Strains show variations in the arrangement of these BGCs that resemble those seen in gerumycin gene clusters. Genome analyses and invasion assays support our hypothesis that vertically transmitted Ps1 and Ps2 strains have antibacterial activity that could help shape the cuticular microbiome. Thus, our work defines the Pseudonocardia species associated with Acromyrmex ants and supports the hypothesis that Pseudonocardia species could provide a valuable source of new antimicrobials.

Regulation of antimycin biosynthesis by the orphan ECF RNA polymerase sigma factor σ (AntA.).

Antimycins are an extended family of depsipeptides that are made by filamentous actinomycete bacteria and were first isolated more than 60 years ago. Recently, antimycins have attracted renewed interest because of their activities against the anti-apoptotic machineries inside human cells which could make them promising anti-cancer compounds. The biosynthetic pathway for antimycins was recently characterised but very little is known about the organisation and regulation of the antimycin (ant) gene cluster. Here we report that the ant gene cluster in Streptomyces albus is organized into four transcriptional units; the antBA, antCDE, antGF and antHIJKLMNO operons. Unusually for secondary metabolite clusters, the antG and antH promoters are regulated by an extracytoplasmic function (ECF) RNA polymerase sigma factor named σ (AntA) which represents a new sub-family of ECF σ factors that is only found in antimycin producing strains. We show that σ (AntA) controls production of the unusual precursor 3-aminosalicylate which is absolutely required for the production of antimycins. σ (AntA) is highly conserved in antimycin producing strains and the -10 and -35 elements at the σ (AntA) regulated antG and antH promoters are also highly conserved suggesting a common mechanism of regulation. We also demonstrate that altering the C-terminal Ala-Ala residues found in all σ (AntA) proteins to Asp-Asp increases expression of the antFG and antGHIJKLMNO operons and we speculate that this Ala-Ala motif may be a signal for the protease ClpXP.

Novel inducers of the envelope stress response BaeSR in Salmonella Typhimurium: BaeR is critically required for tungstate waste disposal.

The RpoE and CpxR regulated envelope stress responses are extremely important for Salmonella Typhimurium to cause infection in a range of hosts. Until now the role for BaeSR in both the Salmonella Typhimurium response to stress and its contribution to infection have not been fully elucidated. Here we demonstrate stationary phase growth, iron and sodium tungstate as novel inducers of the BaeRregulon, with BaeR critically required for Salmonella resistance to sodium tungstate. We show that functional overlap between the resistance nodulation-cell division (RND) multidrug transporters, MdtA, AcrD and AcrB exists for the waste disposal of tungstate from the cell. We also point to a role for enterobactinsiderophores in the protection of enteric organisms from tungstate, akin to the scenario in nitrogen fixing bacteria. Surprisingly, BaeR is the first envelope stress response pathway investigated in S. Typhimurium that is not required for murine typhoid in either ity(S) or ity(R) mouse backgrounds. BaeR is therefore either required for survival in larger mammals such as pigs or calves, an avian host such as chickens, or survival out with the host altogether where Salmonella and related enterics must survive in soil and water.

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.

Enhanced transcription of the Arabidopsis disease resistance genes RPW8.1 and RPW8.2 via a salicylic acid-dependent amplification circuit is required for hypersensitive cell death.

The Arabidopsis disease resistance (R) genes RPW8.1 and RPW8.2 couple the recognition of powdery mildew pathogens of this plant with the subsequent induction of a localized necrosis, or hypersensitive response (HR). The HR restricts the spread of the infection and renders the plant resistant. One-third of Arabidopsis plants transformed with a genomic fragment containing RPW8.1 and RPW8.2 developed spontaneous HR-like lesions (SHL) in the absence of pathogens. We demonstrate that SHL occurs in transgenic lines that contain multiple copies of the transgene and express RPW8.1 and RPW8.2 at high levels. SHL is associated with salicylic acid (SA) accumulation, and at the site of the lesion, there is increased expression of RPW8.1, increased production of H(2)O(2), and increased expression of pathogenesis-related genes. These lesions are physiologically similar to the pathogen-induced HR mediated by RPW8.1 and RPW8.2. Significantly, environmental conditions that suppress SHL suppress the transcription of RPW8.1 and RPW8.2 and also suppress resistance to powdery mildews, even in transgenic lines containing RPW8.1 and RPW8.2 that normally do not express SHL. Furthermore, treatment with SA increases the transcription of RPW8.1 and RPW8.2, induces SHL, and enhances resistance to powdery mildews. We conclude that HR requires the transcription of RPW8.1 and RPW8.2, which is regulated independently of the pathogen by SA-dependent feedback amplification.

Origin and maintenance of a broad-spectrum disease resistance locus in Arabidopsis.

The broad-spectrum mildew resistance genes RPW8.1 and RPW8.2 define a unique type of plant disease resistance (R) gene, and so far homologous sequences have been found in Arabidopsis thaliana only, which suggests a recent origin. In addition to RPW8.1 and RPW8.2, the RPW8 locus contains three homologs of RPW8, HR1, HR2, and HR3, which do not contribute to powdery mildew resistance. To investigate whether RPW8 has originated recently, and if so the processes involved, we have isolated and analyzed the syntenic RPW8 loci from Arabidopsis lyrata, and from Brassica rapa and B. oleracea. The A. lyrata locus contains four genes orthologous to HR1, HR2, HR3, and RPW8.2, respectively. Two syntenic loci have been characterized in Brassica; one locus contains three genes and is present in both B. oleracea and B. rapa, and the other locus contains a single gene and is detected in B. rapa only. The Brassica homologs have highest similarity to HR3. Sequence analyses suggested that the RPW8 gene family in Brassicaceae originated from an HR3-like ancestor gene through a series of duplications and that RPW8.1 and RPW8.2 evolved from functional diversification through positive selection several MYA. Examination of the sequence polymorphism of 32 A. thaliana accessions at the RPW8 locus and their disease reaction phenotypes revealed that the polymorphic RPW8 locus defines a major source of resistance to powdery mildew diseases. A possible evolutionary mechanism by which functional polymorphism at the AtRPW8 locus has been maintained in contemporary populations of A. thaliana is discussed.

COI1 links jasmonate signalling and fertility to the SCF ubiquitin-ligase complex in Arabidopsis.

Jasmonates (JAs) regulate Arabidopsis thaliana wound and defence responses, pollen development, and stress-related growth inhibition. Significantly, each of these responses requires COI1, an F-box protein. Other F-box proteins interact with SKP1 and cullin proteins to form SCF complexes that selectively recruit regulatory proteins targeted for ubiquitination. To determine whether COI1 also functions in an SCF complex, we have characterized Arabidopsis proteins that bind to COI1. An Arabidopsis cDNA expression library was screened in yeast for clones that produce proteins which can bind to COI1. We recovered two SKP1 homologues and a histone deacetylase. The Arabidopsis F-box protein TIR1 interacted with SKP1 proteins, but not with the histone deacetylase. Mutant COI1 proteins revealed that the F-box is required for interaction with SKP1s, but that sequences in leucine-rich repeat domains are required for interaction with the histone deacetylase. Epitope-tagged COI1 was introduced into Arabidopsis plants and cell cultures. Co-immunoprecipitation experiments confirmed the interaction in planta of COI1 with SKP1-like proteins and histone deacetylase, and also indicated that COI1 interacted with cullin. These results suggest that COI1 forms an SCFCOI1 complex in vivo. COI1 is therefore expected to form a functional E3-type ubiquitin ligase in plants and to regulate expression of jasmonate responsive genes, possibly by targeted ubiquitination of a histone deacetylase.