Gene expression profile in response to <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">manihotis</Emphasis> infection in cassava using a cDNA microarray

A cassava cDNA microarray based on a large cassava EST database was constructed and used to study the incompatible interaction between cassava and Xanthomonas axonopodis pv. manihotis (Xam) strain CIO151. For microarray construction, 5700 clones from the cassava unigene set were amplified by polymerase chain reaction (PCR) and printed on glass slides. Microarray hybridization was performed using cDNA from cassava plants (resistant variety MBra685) collected at 12, 24, 48 h and 7 and 15 days post-infection as treatment and cDNA from mock-inoculated plants as control. A total of 199 genes were found to be differentially expressed (126 up-regulated and 73 down-regulated). A greater proportion of differentially-expressed genes was observed at 7 days after inoculation. Expression profiling and cluster analyses indicate that, in response to inoculation with Xam, cassava induces dozens of genes, including principally those involved in oxidative burst, protein degradation and pathogenesis-related (PR) genes. In contrast, genes encoding proteins that are involved in photosynthesis and metabolism were down regulated. In addition, various other genes encoding proteins with unknown function or showing no similarity to other proteins were also induced. Quantitative real time PCR experiments confirmed the reliability of our microarray data. In addition we showed that some genes are induced more rapidly in the resistant than in the susceptible cultivar.These authors made equal contributions to this work.     

Tomato contains two differentially expressed genes encoding B-type phytochromes,neither of which can be considered an ortholog of Arabidopsis phytochrome B

Tomato (Solanum lycopersicon L.) contains two B-type phytochrome genes (PHYB1 and PHYB2). Fragments of these two PHYB were cloned following amplification by the polymerase chain reaction of a portion of their relatively well conserved 5 coding regions. Polypeptides encoded by these gene fragments exhibit 90% sequence identity. These two PHYB are independently expressed in organ-specific fashion. In mature plants, PHYB2 mRNA is most abundant in fruit and PHYB1 mRNA in expanded leaves. A phylogenetic analysis fails to establish which tomato PHYB is orthologous to either Arabidopsis PHYB or PHYD, the latter being a second B-type phytochrome. Instead, this analysis indicates that following the divergence of the Solanaceae and Brassicaceae from one another, a PHYB gene duplicated independently in each lineage. Consequently, Arabidopsis PHYB mutants cannot be considered strictly equivalent to the tomato tri mutants, which appear to be mutated at the PHYB1 locus. Similarly, other putative PHYB mutants might not be equivalent to those described for Arabidopsis and tomato. This situation complicates efforts to determine PHYB function because there might be no one answer to this question.Abbreviations PCR polymerase chain reaction - PHY undesignated phytochrome gene - PHYA, PHYB, etc phytochrome gene(s) of the A, B, etc. type This research was supported by USDA NRICGP grant 93-00939 and by NATO travel grant CRG 931183. It was initiated when two of us (L.H.P., M.-M.C.-P.) spent a sabbatical year at the Institut National de la Recherche Agronomique in Versailles, France. L.H.P. gratefully acknowledges support provided by a senior guest fellowship from the Ministère de l'enseignement superieur et de la recherche during his stay in Versailles. L.H.P. and M.-M.C.-P thank all of their colleagues in Versailles for their warm hospitality and their willingness to share their expertise with us. We also thank Russell Malmberg, Richard Meagher and Robert Price for helpful discussions concerning the interpretation of molecular phylogenies.     

Respiratory variations of aortic VTI: a new index of hypovolemia and fluid responsiveness

In 12 mechanically ventilated and anesthetized rabbits, we investigated whether the magnitude of respiratory changes in the aortic velocity time integral (VTI(Ao)), recorded by transthoracic echocardiography (TTE) during a stepwise blood withdrawal and restitution, could be used as a reliable indicator of volume depletion and responsiveness. At each step, left and right ventricular dimensions and the aortic diameter and VTI(Ao) were recorded to calculate stroke volume (SV) and cardiac output (CO). Respiratory changes of VTI(Ao) (maximal - minimal values divided by their respective means) were calculated. The amount of blood withdrawal correlated negatively with left and right ventricular diastolic diameters, VTI(Ao), SV, and CO and correlated directly with respiratory changes of VTI(Ao). Respiratory VTI(Ao) variations (but not other parameters) at the last blood withdrawal step was also correlated with changes in SV after blood restitution (r = 0.83, P < 0.001). In conclusion, respiratory variations in VTI(Ao) using TTE appear to be a sensitive index of blood volume depletion and restitution. This dynamic parameter predicted fluid responsiveness more reliably than static markers of cardiac preload.     

Opsonizing properties of natural antibodies of rainbow trout, Oncorhynchus mykiss (Walbaum)

In order to confirm previous observations in which a protective effect of rainbow trout natural antibodies against furunculosis was suspected, phagocytosis studies wereconducted in vitro , using combinations of rainbow trout sera with high or low levels of natural antibodies and active or inactivated complement as opsonizing factors. Opsonization was observed in all the cases where complement was present, and to a lesser degree with sera containing only natural antibodies. The results confirm the prime importance of the complement system and provide additional evidence for a possible role of natural antibodies in antimicrobial defences.     

RNA Mimicry by the Fap7 Adenylate Kinase in Ribosome Biogenesis

During biogenesis of the 40S and 60S ribosomal subunits, the pre-40S particles are exported to the cytoplasm prior to final cleavage of the 20S pre-rRNA to mature 18S rRNA. Amongst the factors involved in this maturation step, Fap7 is unusual, as it both interacts with ribosomal protein Rps14 and harbors adenylate kinase activity, a function not usually associated with ribonucleoprotein assembly. Human hFap7 also regulates Cajal body assembly and cell cycle progression via the p53–MDM2 pathway. This work presents the functional and structural characterization of the Fap7–Rps14 complex. We report that Fap7 association blocks the RNA binding surface of Rps14 and, conversely, Rps14 binding inhibits adenylate kinase activity of Fap7. In addition, the affinity of Fap7 for Rps14 is higher with bound ADP, whereas ATP hydrolysis dissociates the complex. These results suggest that Fap7 chaperones Rps14 assembly into pre-40S particles via RNA mimicry in an ATP-dependent manner. Incorporation of Rps14 by Fap7 leads to a structural rearrangement of the platform domain necessary for the pre-rRNA to acquire a cleavage competent conformation.     

Maurocalcine and peptide A stabilize distinct subconductance states of ryanodine receptor type 1, revealing a proportional gating mechanism

Maurocalcine (MCa) isolated from Scorpio maurus palmatus venom shares 82% sequence identity with imperatoxin A. Both scorpion toxins are putative mimics of the II-III loop peptide (termed peptide A (pA)) of alpha(1s)-dihydropyridine receptor and are thought to act at a common site on ryanodine receptor type 1 (RyR1) important for skeletal muscle EC coupling. The relationship between the actions of synthetic MCa (sMCa) and pA on RyR1 were examined. sMCa released Ca(2+) from SR vesicles (EC(50) = 17.5 nm) in a manner inhibited by micromolar ryanodine or ruthenium red. pA (0.5-40 microm) failed to induce SR Ca(2+) release. Rather, pA enhanced Ca(2+) loading into SR and fully inhibited Ca(2+)-, caffeine-, and sMCa-induced Ca(2+) release. The two peptides modified single channel gating behavior in distinct ways. With Cs(+)-carrying current, 10 nm to 1 microm sMCa induced long lived subconductances having 48% of the characteristic full open state and occasional transitions to 29% at either positive or negative holding potentials. In contrast, pA stabilized long lived channel closures with occasional burst transitions to 65% (s1) and 86% (s2) of the full conductance. The actions of pA and sMCa were observed in tandem. sMCa stabilized additional subconductance states proportional to pA-induced subconductances (i.e. 43% of pA-modified s1 and s2 substates), revealing a proportional gating mechanism. [(3)H]Ryanodine binding and surface plasmon resonance analyses indicated that the peptides did not interact by simple competition for a single class of mutually exclusive sites on RyR1 to produce proportional gating. The actions of sMCa were also observed with ryanodine-modified channels and channels deficient in immunophilin 12-kDa FK506-binding protein. These results provide evidence that sMCa and pA stabilize distinct RyR1 channel states through distinct mechanisms that allosterically stabilize gating states having proportional conductance.     

Chemical quenching of singlet oxygen by plastoquinols and their oxidation products in Arabidopsis

Prenylquinols (tocochromanols and plastoquinols) serve as efficient physical and chemical quenchers of singlet oxygen (¹O₂) formed during high light stress in higher plants. Although quenching of ¹O₂ by prenylquinols has been previously studied, direct evidence for chemical quenching of ¹O₂ by plastoquinols and their oxidation products is limited in vivo. In the present study, the role of plastoquinol‐9 (PQH₂‐9) in chemical quenching of ¹O₂ was studied in Arabidopsis thaliana lines overexpressing the SOLANESYL DIPHOSPHATE SYNTHASE 1 gene (SPS1oex) involved in PQH₂‐9 and plastochromanol‐8 biosynthesis. In this work, direct evidence for chemical quenching of ¹O₂ by plastoquinols and their oxidation products is presented, which is obtained by microscopic techniques in vivo. Chemical quenching of ¹O₂ was associated with consumption of PQH₂‐9 and formation of its various oxidized forms. Oxidation of PQH₂‐9 by ¹O₂ leads to plastoquinone‐9 (PQ‐9), which is subsequently oxidized to hydroxyplastoquinone‐9 [PQ(OH)‐9]. We provide here evidence that oxidation of PQ(OH)‐9 by ¹O₂ results in the formation of trihydroxyplastoquinone‐9 [PQ(OH)₃‐9]. It is concluded here that PQH₂‐9 serves as an efficient ¹O₂ chemical quencher in Arabidopsis, and PQ(OH)₃‐9 can be considered as a natural product of ¹O₂ reaction with PQ(OH)‐9. The understanding of the mechanisms underlying ¹O₂ chemical quenching provides information on the role of plastoquinols and their oxidation products in the response of plants to photooxidative stress.