The Arabidopsis AtNPR1 inversely modulates defense responses against fungal, bacterial, or viral pathogens while conferring hypersensitivity to abiotic stresses in transgenic rice

The nonexpressor of pathogenesis-related (PR) genes (NPR1) protein plays an important role in mediating defense responses activated by pathogens in Arabidopsis. In rice, a disease-resistance pathway similar to the Arabidopsis NPR1-mediated signaling pathway one has been described. Here, we show that constitutive expression of the Arabidopsis NPR1 (AtNPR1) gene in rice confers resistance against fungal and bacterial pathogens. AtNPR1 exerts its protective effects against fungal pathogens by priming the expression of salicylic acid (SA)-responsive endogenous genes, such as the PR1b, TLP (PR5), PR10, and PBZ1. However, expression of AtNPR1 in rice has negative effects on viral infections. The AtNPR1-expressing rice plants showed a higher susceptibility to infection by the Rice yellow mottle virus (RYMV) which correlated well with a misregulation of RYMV-responsive genes, including expression of the SA-regulated RNA-dependent RNA polymerase 1 gene (OsRDR1). Moreover, AtNPR1 negatively regulates the expression of genes playing a role in the plant response to salt and drought stress (rab21, salT, and dip1), which results in a higher sensitivity of AtNPR1 rice to the two types of abiotic stress. These observations suggest that AtNPR1 has both positive and negative regulatory roles in mediating defense responses against biotic and abiotic stresses.     

High-throughput microplate assay for the determination of drug partition coefficients

Partition coefficients (K(p)) of drugs between the phospholipid bilayer and the aqueous phase provide useful information in quantitative structure-activity relationship studies. Hexadecylphosphocholine (HePC) micelles, composed of a zwitterionic hydrophilic surface and a hydrophobic core, mimic the biomembranes and have several advantages over other lipid structures to assess K(p) values. Their preparation is easy, fast and avoids the use of toxic organic solvents, and the output has fewer spectroscopic interferences. Here, we describe a high-throughput microplate protocol for assessing the K(p) of drugs using HePC micelles as membrane models and derivative spectrophotometry as the detection technique. Moreover, the time-consuming data treatment to assess K(p) values is easily performed by a dedicated Excel routine developed here and described in detail. The K(p) values of nonsteroidal anti-inflammatory drugs (acemetacin, clonixin, diclofenac and indomethacin) were determined to show the simplicity of the method and to validate this protocol, which provides K(p) values (n = 3) of two drugs in ～ 2 h.     

A potato carboxypeptidase inhibitor gene provides pathogen resistance in transgenic rice

A defensive role against insect attack has been traditionally attributed to plant protease inhibitors. Here, evidence is described of the potential of a plant protease inhibitor, the potato carboxypeptidase inhibitor (PCI), to provide resistance to fungal pathogens when expressed in rice as a heterologous protein. It is shown that rice plants constitutively expressing the pci gene exhibit resistance against the economically important pathogens Magnaporthe oryzae and Fusarium verticillioides . A M. oryzae carboxypeptidase was purified by affinity chromatography and further characterized by mass spectrometry. This fungal carboxypeptidase was found to be a novel carboxypeptidase B which was fully inhibited by PCI. Overall, the results indicate that PCI exerts its antifungal activity through the inhibition of this particular fungal carboxypeptidase B. Although pci confers protection against fungal pathogens in transgenic rice, a significant cost in insect resistance is observed. Thus, the weight gain of larvae of the specialist insect Chilo suppressalis (striped stem borer) and the polyphagous insect Spodoptera littoralis (Egyptian cotton worm) fed on pci rice is significantly larger than that of insects fed on wild-type plants. Homology-based modelling revealed structural similarities between the predicted structure of the M. oryzae carboxypeptidase B and the crystal structure of insect carboxypeptidases, indicating that PCI may function not only as an inhibitor of fungal carboxypeptidases, but also as an inhibitor of insect carboxypeptidases. The potential impact of the pci gene in terms of protection against fungal and insect diseases is discussed.     

Fungus- and wound-induced accumulation of mRNA containing a class II chitinase of the pathogenesis-related protein 4 (PR-4) family of maize

Pathogenesis-related (PR) proteins are plant proteins that are induced in response to pathogen attack. PR proteins are grouped into independent families based on their sequences and properties. The PR-4 family comprises class I and class II chitinases. We have isolated a full-length cDNA encoding a chitinase from maize which shares a high degree of nucleotide and amino acid sequence homology with the class II chitinases of the PR-4 family of PR proteins. Our results indicate that fungal infection, and treatment either with fungal elicitors or with moniliformin, a mycotoxin produced by the fungus Fusarium moniliforme, increase the level of ZmPR4 mRNA. In situ mRNA hybridization analysis in sections obtained from fungus-infected germinating embryos revealed that ZmPR4 mRNA accumulation occurs in those cell types that first establish contact with the pathogen. ZmPR4 mRNA accumulation is also stimulated by treatment with silver nitrate whereas the application of the hormones gibberellic acid or acetylsalicylic acid has no effect. Wounding, or treatment with abscisic acid or methyl jasmonate, results in accumulation of ZmPR4 mRNA in maize leaves. Furthermore, the ZmPR4 protein was expressed in Escherichia coli, purified and used to obtain polyclonal antibodies that specifically recognized ZmPR4 in protein extracts from fungus-infected embryos. Accumulation of ZmPR4 mRNA in fungus-infected maize tissues was accompanied by a significant accumulation of the corresponding protein. The possible implications of these findings as part of the general defence response of maize plants against pathogens are discussed.     

Primary structure of the activation segment of procarboxypeptidase A from porcine pancreas

The complete primary structure of the activation segment of monomeric procarboxypeptidase A from porcine pancreas has been determined by automated and manual Edman-like degradation methods performed on its fragments generated by enzymatic cleavage. The polypeptide consists of 94 residues, with a molecular mass of 10,768, and presents a high proportion of acidic and hydrophobic residues and a proline-rich region in the center of the molecule. Comparison of this sequence with the already reported equivalent sequence deduced from rat procarboxypeptidase A cDNA reveals a very high degree of homology between the two propeptides (up to a 81% of identities), which is even higher in certain large zones of the molecule.     

A model of excitatory synaptic interactions between pacemakers. Its reality,its generality,and the principles involved

This is a model of the steady-state influence of one pacemaker neuron upon another across a synapse with EPSP's. Its postulates require firstly the spontaneous regularity of both cells, whose intervals are E and N, respectively. In addition, they require a special shortening or negative delay of the interspike interval by one or more EPSP's, with a V-shaped dependence of the delay on the position or phase of the EPSP's in the interval; the minimum of the delay function corresponds to the earliest EPSP arrival phase () that triggers a spike immediately. Finally, they impose on the variables certain bounds. The model's behavior has two main features. The first is a zig-zag relationship with an overall increasing trend between the steady-state pre- and post-synaptic discharge intensities (Fig. 7). The zig-zag is formed predominantly, if not exclusively, by segments with positive slopes that are rational fractions. Passage from one such segment to others is negatively-sloped (paradoxical), involving staggered positively-sloped segments whose details are unclear for weak presynaptic discharges and discontinuities for intense discharges. The same postsynaptic intensity may result from several presynaptic ones; the maximum postsynaptic intensity may reflect refractoriness, or the earliest instants of immediate triggering. The second main feature is the locking of the discharges in an invariant forward and backward temporal relation. With at most one EPSP per postsynaptic spike, locking is always present. If the presynaptic interval E is in the closed {rN+,(r+1)N} range, locking is 1:r+1, either stable at a greater-than- phase or unstable at a smaller one; arrivals at integral multiples of N do not affect the postsynaptic intensity. If E is in {rN, rN+} (r>0), locking is at other ratios (e.g., 2:3) and less apparent. With more than one EPSP per spike, when E is below bounds that depend on the interspike interval and the point of earliest triggering, locking happens in the simple s:1 ratio (s=2,3, ...) and is stable; when E is above those bounds, there are E ranges where locking is in other ratios (e.g., 3:2) and ranges where behavior is unclear. The validity of any model is based jointly upon an a priori judgment as to whether postulates depart reasonably little from nature, and upon an a posteriori experimental comparison of modelled and real behaviors. The model's domain of applicability depends on the specific embodiment, each of the latter tolerating characteristically each departure. The present model will be evaluated in the crayfish stretch-receptor neuron (Diez-Martínez et al., in preparation). The model is applicable to any physical system that complies with its postulates, and evidence compatible with this notion is available in many disparate fields. It illustrates the modelling path to a scientific proposition, other paths being inference from experimentation, or deduction from premises acceptable at other approach levels (in this case, for example, from that of synaptic mechanisms). The periodicity postulates set this model within the category of those for oscillators. The notion of an oscillator has a far broader applicability than appears at first sight, since all physically realizable systems have some predominant output frequency, i.e., to a certain extent are oscillators.Supported by funds from the Brain Research Institute, UCLASupported by FAPESP (Sao Paulo, Brazil). Present address: Esc. Politecnica, Dee, University of Sao Paulo, Cid. Univ., CP 8174 Sao Paulo, S.P., Brazil     

Presynaptic irregularity and pacemaker inhibition

It is known (e.g., Perkel et al., 1964) that when a pacemaker neuron elicits IPSP's in another, there are domains called paradoxical segments where in the steady-state i) faster inhibitory discharges determine faster inhibited ones, and ii) pre- and postsynaptic spikes are locked in an invariant forward-and-backward positioning in time, spikes alternating in the ratios 1:1 (1 pere for 1 postsynaptic), 1:2, 2:1..., that are also the slopes of the synaptic rate-transformation. The present project examined the matter further in the inhibitory synapse upon the crayfish tonic stretch receptor neuron, confirming the above. In addition it showed that locking and alternation existed also in the segments interposed between the 1:2, 1:1 and 2:1 paradoxical segments, even though they were not as marked and apparent, and that when tests were close to each other their order became influential and hysteresis-like phenomena appeared. The main finding was that paradoxical rate-relations, locking and alternation persisted when the presynaptic train was irregularized up to interval coefficients of variation of around 0.20 (Figs. 2–5). Therefore, both phenomena may not simply be laboratory curiosities, but also have a role in natural operation where probably a substantial population of neurons exhibits that kind of irregularity. As presynaptic irregularity increased, the paradoxical segment slopes and widths decreased and locking and alternation became less clear-cut. With CV's of about 0.20, only a relatively narrow 1:1 paradoxical segment with about O slope and little locking and alternation remained (Figs. 2b, 3g, 4right, 5third row). With larger CV's, the rate relation decreased monotonically and there was no locking nor alternation (Figs. 2e, 3h, 5bottom row). The postsynaptic discharge was more regular and had fewer changes in the number of presynaptic spikes per post-synaptic interval within paradoxical segments (particularly in their centers) than in segments interposed between them (left vs. right-hand columns in Figs. 5, 6; Fig. 7): the contrast, remarkable for regular stimuli, attenuated as variability increased. The following conclusions are relevant to coding of spike trains across a synapse with IPSP's. i) With fairly regular discharges, the same postsynaptic rate may result from several presynaptic ones (e.g., may result from rates in the 1:1 and 2:1 paradoxical segments and in the interposed one, Fig.2): in some cases but not others, the precise presynaptic rate can be identified on the basis of postsynaptic CV's, interval histograms and cycle slips. ii) A small rate change in a regular presynaptic discharge will have very different postsynaptic consequences depending on where it happens: if across a paradoxical-interposed boundary, for instance, it will cause remarkable rate, pattern and correlation changes. iii) The trans-synaptic mapping of variability involves an increase for the more regular presynaptic discharges and a decrease for the more irregular ones. iv) The postsynaptic discharge was slower with IPSP's than without in most cases; however, when the control discharge was weak or absent, IPSP's accelerated it. Results are relevant also to the operation of periodically performing systems that involve neuronal correlates, indicating that it is necessary in every case to ask whether zigzag relations and locking occur. The delay function plots the arrival time of an IPSP (or IPSP burst) relative to the last postsynaptic spike, i.e., the phase ( in Fig. 1b), against the interval lengthening produced, i.e., the delay (). In all cases, most points clustered around a straight line (Fig. 8), whose slope and ordinate intercept were in the 0.43–0.87 and the 0.02–0.52 ranges, respectively, for single IPSP's. The slope reflects how the IPSP effectiveness depends on when it arrives in the cycle; the intercept reflects the IPSP effectiveness. Large phases often showed aberrant points whose ordinates were either large (and having special formal implications), or very small (perhaps reflecting conduction and synaptic delays), or clustered around a second straight segment with a large negative slope (when spontaneous rates were low) (Fig. 8c). Delay functions for widely separated pairs of IPSP's could be multi-valued, points clustering around 2 or 3 parallel straight lines. A mathematical model of pacemaker inhibitory synaptic interactions (Segundo, 1979) agreed with this embodiment insofar as some postulated properties are concerned (e.g., regular discharge, interval lengthening by IPSP's, linear delay functions with slopes around 0.7) and as to the main aspects of the preparation's behavior (i.e., zigzag rate relations and locking), but not in terms of some aspects of the postulates (e.g., interval variability, rebound) or behavior (e.g., segment boundaries, jitter in the locking, and hysteresis). The model was judged to be on the balance satisfactorily realistic.Supported by funds from the Brain Research Institute, UclaSupported by FAPESP (Sao Paulo, Brazil)     

Statistical Analysis of Membrane Potential Fluctuations: Relation with Presynaptic Spike Train

In a study of integration at the single neuron level, the relationships between the postsynaptic membrane potential and the presynaptic spike train were analyzed. Fluctuations in membrane potential of neurons in the visceral ganglion of Aplysia were measured and described by histograms. The histogram estimates the probability density function of the membrane potential. Comparisons were made among histograms when there was no synaptic input, and when there was a single input in which variations were made in the PSP (postsynaptic potential) sign, i.e. excitatory or inhibitory, and arrival statistics, e.g. slow or fast, regular, Poisson-like, or patterned. This was examined in cells where the membrane potential was constant and in cells in which there was spontaneous pacemaker activity. The form of the histogram depended on whether the neuron was spontaneously quiescent or a pacemaker, or whether it received presynaptic input and, if it did, on the sign and temporal characteristics of such input. From such histograms the mean firing rate of output spike trains can be predicted; additional information of a temporal nature is required, however, to predict features of the interval structure of the output train. Suggestions are made concerning the way the nervous system might utilize the information summarized in the membrane potential histogram.     

Input-output relations in computer-simulated nerve cells

Summary This communication examines, in digital computer simulated networks, the input-output relation established at synaptic level. It is restricted to excitatory junctions and analyzes the changes in post-synaptic discharge which occur when the number of pre-synaptic terminals increases while the EPSP size decreases, when the statistical structure or form (as measured by the interspike interval mean, standard deviation, histogram and by the autocorrelogram) of the spike train in each pre-synaptic fiber changes, and/or when the interdependence between pre-synaptic fibers varies from complete independence to strong dependence.1Independent Pre-synaptic Terminals. When the number of pre-synaptic terminals increases and the EPSP size decreases proportionally (while the input form remains constant), the post-synaptic interspike interval mean increases slightly, the standard deviation decreases markedly, the histogram becomes sharp and narrow and the autocorrelogram becomes periodic. When, on the other hand, the pre-synaptic form varies (while the number of terminals and the EPSP size remain constant), the effect upon the post-synaptio output depends upon the given number of terminals and EPSP size. If terminals are few and EPSP's large, the output varies with the pre-synaptic form. The post-synaptic coefficient of variation is linearly related to the pre-synaptic coefficient of variation, the slope decreasing as the number of inputs increases. If terminals are numerous and weak, the pre-synaptic form ceases to be influential and the post-synaptic cell generates the same output regardless of the detailed structure of the corresponding input. The output common to any combination of independent weak input forms is a very regular train of evenly spaced spikes. (This conclusion is valid unless pre-synaptic terminals fire at extremely low rates.) Such results are mathematically predictable in a simple and realistic model of membrane potential and threshold dynamics (see Appendix).As the EPSP size increases, all other variables being equal, the post-synaptic interval mean decreases monotonically. The decrease is smooth or in steps depending on whether the pre-synaptic form is Poisson or pacemaker, respectively. Post-synaptic spikes are effectively blocked by relatively small numbers of inhibitory terminals.2Dependent Pre-synaptic Terminals. When there is a physiological amount of interdependence between the presynaptic terminals that impinge upon the post-synaptic cell, the activity of the latter is a function of the statistical form of the input channels, even when the latter are numerous and weak. This happens when interdependence involves only a proportion of all terminals or only the terminals within separate and independent groups.In order to understand the transactions that take place in the nervous system, it is necessary to identify the presynaptic statistics that influence the corresponding post-synaptic discharge. When pre-synaptic terminals produce large PSP's their influence is dominant and exerted by way of the precise statistical form of the discharge. When terminals produce small PSP's their influence is contingent on their degree of interdependence. If they are uncorrelated, they act exclusively by way of their mean rates and provide a smooth adjustment of the post-synaptic membrane potential and firing rate. If terminals are correlated, they act by way of several statistical features and assume a dominant role that determines a precise relation between pre-synaptic timing and post-synaptic firing. The degree of inter-terminal correlation is thus a functionally significant variable.

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Zusammenfassung Mit Hilfe eines Digitalrechners wurden die Eingangs-und Ausgangsbeziehungen auf synaptischer Ebene untersucht und dargestellt. Diese Untersuchung erstreckt sich auf erregende Synapsen und analysiert die Veränderungen postsynaptischer Aktionspotentiale, die auftreten, 1. wenn die Anzahl der präsynaptischen axonischen Endigungen ansteigt, während andererseits die Amplitude des EPSP abnimmt; 2. wenn sich die statistische Struktur oder Form der Spike-Kette in jeder präsynaptischen Faser verändert; und/oder 3. wenn die Beziehungen zwischen den präsynaptischen Fasern von völliger Unabhängigkeit bis zu starker Abhängigkeit variiert werden.1Unabhängige präsynaptische Endigungen. Mit zunehmender Anzahl präsynaptischer Endigungen bei gleichzeitiger proportionaler Abnahme des EPSP (Input Form konstant) treten folgende Veränderungen auf: a) das durchschnittliche Intervall zwischen den postsynaptischen Spikes nimmt etwas zu; b) die mittlere statistische Abweichung (standard deviation) nimmt erheblich ab; c) die Form des Histogramms wird eng umschrieben; und d) das Autokorrelogramm nimmt periodischen Charakter an. Wenn andererseits die präsynaptische Form verändert wird (während die Anzahl der Endigungen sowie die Größe des EPSP konstant bleibt), hängt der am postsynaptischen Ausgang registrierte Effekt von der gegebenen Anzahl der Endigungen und von der Größe des EPSP ab. Ist die Anzahl der Endigungen gering und das EPSP groß, dann variiert der Ausgang mit der präsynaptischen Form. Der postsynaptische Variationskoeffizient steht dann in linearer Abhängigkeit vom präsynaptischen Variationskoeffizienten, wobei die Steigung der Geraden mit zunehmendem Eingang abnimmt. Sind die Endigungen zahlreich und die Größen der EPSPs gering, dann übt die präsynaptische Form keinen Einfluß mehr aus, und das von der postsynaptischen Zelle erzeugte Ausgangsprodukt wird unabhängig von der detaillierten Struktur des Eingangs. Für eine jegliche Kombination von unabhängigen und schwachen Eingangsformen stellt sich das Ausgangsprodukt in Form einer sehr regelmäßig gestalteten und durch gleichmäßige Abstände gekennzeichneten Spike-Kette dar (diese Folgerung gilt nur für die Fälle, in denen die präsynaptischen Endigungen sich nicht äußerst langsam entladen). Diese Resultate können mathematisch an Hand eines einfachen Membranmodells abgeleitet werden (s. Appendix).Wenn das EPSP in Größe ansteigt, alle anderen Variablen jedoch gleich bleiben, dann nimmt das postsynaptische Intervall fortwährend ab. Dieser Abfall ist entweder gleichmäßig (präsynaptische Form: Poisson) oder stufenweise (präsynaptische Form: pacemaker). Die postsynaptischen Aktionspotentiale werden durch eine vergleichsweise kleine Anzahl von hemmenden Endigungen wirkungsvoll blockiert.2Abhängige präsynaptische Endigungen. Wenn sich der Grad der Abhängigkeit zwischen den präsynaptischen Endigungen in physiologischen Grenzen bewegt, dann kann die Aktivität der postsynaptischen Zelle als eine Funktion der statistischen Form der Eingangskanäle angegeben werden, und das sogar, wenn die letzteren zahlreich und schwach sind. Dieser Fall tritt dann ein, wenn die Abhängigkeit zwischen den präsynaptischen Endigungen nur einen Teil aller Endigungen oder nur die Endigungen innerhalb getrennter und unabhängiger Gruppen betrifft.Um die im Nervensystem stattfindenden Übertragungen zu verstehen, ist es notwendig, diejenigen präsynaptischen Statistiken zu idendifizieren, die entsprechende postsynaptische Entladungen beeinflussen. Wenn präsynaptische Endigungen große PSPs hervorrufen, dann ist ihr Einfluß dominierend und wird entsprechend der präzisen statistischen Form ausgeübt. Wenn die Endigungen kleine PSPs hervorrufen, dann hängt ihr Einfluß weitgehend von dem Grad der Abhängigkeit voneinander ab. Wenn die präsynaptischen Endigungen unkorreliert sind, dann vermitteln ihre durchschnittlichen, präsynaptischen Entladungsgeschwindigkeiten eine gleichmäßige Regulierung des postsynaptischen Membranpotentials und der postsynaptischen Entladungsgeschwindigkeiten. Sind andererseits die präsynaptischen Endigungen korreliert, dann nehmen sie eine dominierende Funktion ein, und die Beziehungen zwischen präsynaptischer Regulierung und postsynaptischer Entladung können präzise definiert werden. Somit stellt sich der Grad der Abhängigkeit zwischen den präsynaptischen Endigungen als eine funktionell bedeutende Variable dar.

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Supported by a Research Career Program (J. P. S.) and by Grants from the USPHS (NB-02501, NB-05264, NB-07325), and by Air Force Project Rand. Computing assistance was obtained from the UCLA Health Science Computing Facility sponsored by NIH Grant FR-3, and from the Brain Research Institute Data Processing Laboratory.