Host-secreted antimicrobial peptide enforces symbiotic selectivity in Medicago truncatula.

Legumes engage in root nodule symbioses with nitrogen-fixing soil bacteria known as rhizobia. In nodule cells, bacteria are enclosed in membrane-bound vesicles called symbiosomes and differentiate into bacteroids that are capable of converting atmospheric nitrogen into ammonia. Bacteroid differentiation and prolonged intracellular survival are essential for development of functional nodules. However, in the Medicago truncatula-Sinorhizobium meliloti symbiosis, incompatibility between symbiotic partners frequently occurs, leading to the formation of infected nodules defective in nitrogen fixation (Fix-). Here, we report the identification and cloning of the M. truncatula NFS2 gene that regulates this type of specificity pertaining to S. meliloti strain Rm41. We demonstrate that NFS2 encodes a nodule-specific cysteine-rich (NCR) peptide that acts to promote bacterial lysis after differentiation. The negative role of NFS2 in symbiosis is contingent on host genetic background and can be counteracted by other genes encoded by the host. This work extends the paradigm of NCR function to include the negative regulation of symbiotic persistence in host-strain interactions. Our data suggest that NCR peptides are host determinants of symbiotic specificity in M. truncatula and possibly in closely related legumes that form indeterminate nodules in which bacterial symbionts undergo terminal differentiation.

UV-B-responsive association of the Arabidopsis bZIP transcription factor ELONGATED HYPOCOTYL5 with target genes, including its own promoter.

In plants subjected to UV-B radiation, responses are activated that minimize damage caused by UV-B. The bZIP transcription factor ELONGATED HYPOCOTYL5 (HY5) acts downstream of the UV-B photoreceptor UV RESISTANCE LOCUS8 (UVR8) and promotes UV-B-induced photomorphogenesis and acclimation. Expression of HY5 is induced by UV-B; however, the transcription factor(s) that regulate HY5 transcription in response to UV-B and the impact of UV-B on the association of HY5 with its target promoters are currently unclear. Here, we show that HY5 binding to the promoters of UV-B-responsive genes is enhanced by UV-B in a UVR8-dependent manner in Arabidopsis thaliana. In agreement, overexpression of REPRESSOR OF UV-B PHOTOMORPHOGENESIS2, a negative regulator of UVR8 function, blocks UV-B-responsive HY5 enrichment at target promoters. Moreover, we have identified a T/G-box in the HY5 promoter that is required for its UV-B responsiveness. We show that HY5 and its homolog HYH bind to the T/G(HY5)-box cis-acting element and that they act redundantly in the induction of HY5 expression upon UV-B exposure. Therefore, HY5 is enriched at target promoters in response to UV-B in a UVR8 photoreceptor-dependent manner, and HY5 and HYH interact directly with a T/G-box cis-acting element of the HY5 promoter, mediating the transcriptional activation of HY5 in response to UV-B.

The circadian clock-associated small GTPase LIGHT INSENSITIVE PERIOD1 suppresses light-controlled endoreplication and affects tolerance to salt stress in Arabidopsis.

Circadian clocks are biochemical timers regulating many physiological and molecular processes according to the day/night cycle. The small GTPase LIGHT INSENSITIVE PERIOD1 (LIP1) is a circadian clock-associated protein that regulates light input to the clock. In the absence of LIP1, the effect of light on free-running period length is much reduced. Here, we show that in addition to suppressing red and blue light-mediated photomorphogenesis, LIP1 is also required for light-controlled inhibition of endoreplication and tolerance to salt stress in Arabidopsis (Arabidopsis thaliana). We demonstrate that in the processes of endoreplication and photomorphogenesis, LIP1 acts downstream of the red and blue light photoreceptors phytochrome B and cryptochromes. Manipulation of the subcellular distribution of LIP1 revealed that the circadian function of LIP1 requires nuclear localization of the protein. Our data collectively suggest that LIP1 influences several signaling cascades and that its role in the entrainment of the circadian clock is independent from the other pleiotropic effects. Since these functions of LIP1 are important for the early stages of development or under conditions normally experienced by germinating seedlings, we suggest that LIP1 is a regulator of seedling establishment.

LIP1 Regulates the Plant Circadian Oscillator by Modulating the Function of the Clock Component GIGANTEA.

Circadian clocks are biochemical timers regulating many physiological and molecular processes according to the day/night cycles. The function of the oscillator relies on negative transcriptional/translational feedback loops operated by the so-called clock genes and the encoded clock proteins. Previously, we identified the small GTPase LIGHT INSENSITIVE PERIOD 1 (LIP1) as a circadian-clock-associated protein that regulates light input to the clock in the model plant Arabidopsis thaliana. We showed that LIP1 is also required for suppressing red and blue light-mediated photomorphogenesis, pavement cell shape determination and tolerance to salt stress. Here, we demonstrate that LIP1 is present in a complex of clock proteins GIGANTEA (GI), ZEITLUPE (ZTL) and TIMING OF CAB 1 (TOC1). LIP1 participates in this complex via GUANINE EX-CHANGE FACTOR 7. Analysis of genetic interactions proved that LIP1 affects the oscillator via modulating the function of GI. We show that LIP1 and GI independently and additively regulate photomorphogenesis and salt stress responses, whereas controlling cell shape and photoperiodic flowering are not shared functions of LIP1 and GI. Collectively, our results suggest that LIP1 affects a specific function of GI, possibly by altering binding of GI to downstream signalling components.

Functional analysis of amino-terminal domains of the photoreceptor phytochrome B.

At the core of the circadian network in Arabidopsis (Arabidopsis thaliana), clock genes/proteins form multiple transcriptional/translational negative feedback loops and generate a basic approximately 24-h oscillation, which provides daily regulation for a wide range of processes. This temporal organization enhances the fitness of plants only if it corresponds to the natural day/night cycles. Light, absorbed by photoreceptors, is the most effective signal in synchronizing the oscillator to environmental cycles. Phytochrome B (PHYB) is the major red/far-red light-absorbing phytochrome receptor in light-grown plants. Besides modulating the pace and phase of the circadian clock, PHYB controls photomorphogenesis and delays flowering. It has been demonstrated that the nuclear-localized amino-terminal domain of PHYB is capable of controlling photomorphogenesis and, partly, flowering. Here, we show (1) that PHYB derivatives containing 651 or 450 amino acid residues of the amino-terminal domains are functional in mediating red light signaling to the clock, (2) that circadian entrainment is a nuclear function of PHYB, and (3) that a 410-amino acid amino-terminal fragment does not possess any functions of PHYB due to impaired chromophore binding. However, we provide evidence that the carboxyl-terminal domain is required to mediate entrainment in white light, suggesting a role for this domain in integrating red and blue light signaling to the clock. Moreover, careful analysis of the circadian phenotype of phyB-9 indicates that PHYB provides light signaling for different regulatory loops of the circadian oscillator in a different manner, which results in an apparent decoupling of the loops in the absence of PHYB under specific light conditions.

Purification and preliminary characterization of a cold-adapted extracellular proteinase from Trichoderma atroviride.

Eleven cold-tolerant Trichoderma isolates were screened for the production of proteolytic activities at 10 degrees C. Based on the activity profiles determined with paranitroanilide substrates at 5 degrees C, strain T221 identified as Trichoderma atroviride was selected for further investigations. The culture broth of the strain grown at 10 degrees C in casein-containing culture medium was concentrated by lyophilization and subjected to gel filtration, which was followed by chromatofocusing of the fraction showing the highest activity on N-benzoyl-Phe-Val-Arg-paranitroanilide. The purified enzyme had a molecular weight of 24 kDa, an isoelectric point of 7.3 and a pH optimum of 6.2. The temperature optimum of 25 degrees C and the low thermal stability suggested that it is a true cold-adapted enzyme. Substrate specificity data indicate that the enzyme is a proteinase with a preference for Arg or Lys at the P1 position. The effect of proteinase inhibitors suggests that the enzyme has a binding pocket similar to the one present in trypsin.

Phytochrome A-specific signaling in Arabidopsis thaliana.

Among the five phytochromes in Arabidopsis thaliana, phytochrome A (phyA) plays a major role in seedling de-etiolation. Until now more then ten positive and some negative components acting downstream of phyA have been identified. However, their site of action and hierarchical relationships are not completely understood yet.

A switchable light-input, light-output system modelled and constructed in yeast.

BACKGROUND: Advances in synthetic biology will require spatio-temporal regulation of biological processes in heterologous host cells. We develop a light-switchable, two-hybrid interaction in yeast, based upon the Arabidopsis proteins PHYTOCHROME A and FAR-RED ELONGATED HYPOCOTYL 1-LIKE. Light input to this regulatory module allows dynamic control of a light-emitting LUCIFERASE reporter gene, which we detect by real-time imaging of yeast colonies on solid media. RESULTS: The reversible activation of the phytochrome by red light, and its inactivation by far-red light, is retained. We use this quantitative readout to construct a mathematical model that matches the system's behaviour and predicts the molecular targets for future manipulation. CONCLUSION: Our model, methods and materials together constitute a novel system for a eukaryotic host with the potential to convert a dynamic pattern of light input into a predictable gene expression response. This system could be applied for the regulation of genetic networks - both known and synthetic.