Validation of molecular markers linked to apospory in tetraploid races of bahiagrass, Paspalum notatum Flüggé

Tetraploid (2n = 4x = 40) races of Paspalum notatum Flüggé are important natural forage grasses for the tropical and subtropical areas of the Americas. Almost all natural accessions reproduce by obligate aposporous apomixis. Previous work on the species allowed the identification of several molecular markers completely linked to apospory, one component of apomictic reproduction. Moreover, after a fingerprinting characterization of a germplasm collection, 11 amplified fragment length polymorphism (AFLP) markers exclusive to apomictic accessions were detected. The objectives of this work were (1) to validate the presence of molecular markers linked to apospory in tetraploid races of different geographic origins, (2) to determine if markers specific to apomictic accessions were associated with the mode of reproduction, and (3) to develop single-locus markers of apospory that can be used for marker-assisted selection. Thirteen natural apomictic accessions were analyzed. Moreover, the parental plants Q4188 (non-aposporous) and Q4117 (aposporous) and 44 F1 progenies (36 non-aposporous, 8 aposporous) derived from them were used as a validation population. Nine markers [two random amplification of polymorphic DNA (RAPD) and seven AFLP] 100% linked to apospory in Q4117 were tested. Amplification reactions with the corresponding primers showed that all markers were present in the 13 aposporous (apomictic) accessions, but were absent in the non-aposporous controls. On the other hand, linkage analysis of the 11 AFLP markers specific to the apomictic accessions showed that all of them were linked in coupling to apospory (r = 0.00, LOD 13.245). Based on one AFLP (E36M37c), two sequence characterized amplification region (SCAR) markers (SPNA1 and SPNA2) co-segregating with the trait and present in the 13 apomictic accessions were developed. The presence of markers associated with apospory was conserved among tetraploid accessions of different geographic origins. Moreover, the single-locus markers SPNA1 and SPNA2 could be used for routine marker-assisted selection in hybrid populations segregating for apospory and to facilitate the isolation of apospory-related genes.     

An electrostatic switch displaces phosphatidylinositol phosphate kinases from the membrane during phagocytosis

Plasmalemmal phosphatidylinositol (PI) 4,5-bisphosphate (PI4,5P₂) synthesized by PI 4-phosphate (PI4P) 5-kinase (PIP5K) is key to the polymerization of actin that drives chemotaxis and phagocytosis. We investigated the means whereby PIP5K is targeted to the membrane and its fate during phagosome formation. Homology modeling revealed that all PIP5K isoforms feature a positively charged face. Together with the substrate-binding loop, this polycationic surface is proposed to constitute a coincidence detector that targets PIP5Ks to the plasmalemma. Accordingly, manipulation of the surface charge displaced PIP5Ks from the plasma membrane. During particle engulfment, PIP5Ks detached from forming phagosomes as the surface charge at these sites decreased. Precluding the change in surface charge caused the PIP5Ks to remain associated with the phagosomal cup. Chemically induced retention of PIP5K-γ prevented the disappearance of PI4,5P₂ and aborted phagosome formation. We conclude that a bistable electrostatic switch mechanism regulates the association/dissociation of PIP5Ks from the membrane during phagocytosis and likely other processes.     

A critical step for postsynaptic F‐actin organization: Regulation of Baz/Par‐3 localization by aPKC and PTEN

Actin remodeling has emerged as a critical process during synapse development and plasticity. Thus, understanding the regulatory mechanisms controlling actin organization at synapses is exceedingly important. Here, we used the highly plastic Drosophila neuromuscular junction (NMJ) to understand mechanisms of actin remodeling at postsynaptic sites. Previous studies have suggested that the actin‐binding proteins Spectrin and Coracle play a critical role in NMJ development and the anchoring of glutamate receptors most likely through actin regulation. Here, we show that an additional determinant of actin organization at the postsynaptic region is the PDZ protein Baz/Par‐3. Decreasing Baz levels in postsynaptic muscles has dramatic consequences for the size of F‐actin and spectrin domains at the postsynaptic region. In turn, proper localization of Baz at this site depends on both phosphorylation and dephosphorylation events. Baz phosphorylation by its binding partner, atypical protein kinase C (aPKC), is required for normal Baz targeting to the postsynaptic region. However, the retention of Baz at this site depends on its dephosphorylation mediated by the lipid and protein phosphatase PTEN. Misregulation of the phosphorylation state of Baz by genetic alterations in PTEN or aPKC activity has detrimental consequences for postsynaptic F‐actin and spectrin localization, synaptic growth, and receptor localization. Our results provide a novel mechanism of postsynaptic actin regulation through Baz, governed by the antagonistic actions of aPKC and PTEN. Given the conservation of these proteins from worms to mammals, these results are likely to provide new insight into actin organization pathways. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Synaptic targeting domains of synapsin I revealed by transgenic expression in photoreceptor cells.

Synapsins are abundant nerve terminal proteins present at all synapses except for ribbon synapses, e.g. photoreceptor cell synapses. Multiple functions have been proposed for synapsins, including clustering of synaptic vesicles and regulation of synaptic vesicle exocytosis. To investigate the physiological functions of synapsin and to ascertain which domains of synapsin are involved in synaptic targeting in vivo, we expressed synapsin Ib and its N- and C-terminal domains in the photoreceptor cells of transgenic mice. In these cells synapsin Ib is targeted efficiently to synaptic vesicles but has no significant effect on the development, structure or physiology of the synapses. This suggests that synapsin I does not have dominant physiological or morphoregulatory functions at these synapses. Full-length synapsin Ib and the N-terminal domains of synapsin Ib but not its C-terminal domains are transported to synapses, revealing that the molecular apparatus for synaptic targeting of synapsins is also present in cells which form ribbon synapses that normally lack synapsins. This apparatus appears to utilize the conserved N-terminal domains that are shared between all synapsins.     

Cloning and DNA sequencing of the dextranase inhibitor gene (dei) from Streptococcus sobrinus.

Some dextranase-deficient (Dex-) mutants of Streptococcus sobrinus UAB66 (serotype g) synthesize a substance which inhibits dextranase activity (S.-Y. Wanda, A. Camilli, H. M. Murchison, and R. Curtiss III, J. Bacteriol. 176:7206-7212, 1994). This substance produced by the Dex- mutant UAB108 was designated dextranase inhibitor (Dei) and identified as a protein. The Dei gene (dei) from UAB108 has been cloned into pACYC184 to yield pYA2651, which was then used to generate several subclones (pYA2653 to pYA2657). The DNA sequence of dei was determined by using Tn5seq1 transposon mutagenesis of pYA2653. The open reading frame of dei is 990 bp long. It encodes a signal peptide of 38 amino acids and a mature Dei protein of 292 amino acids with a molecular weight of 31,372. The deduced amino acid sequence of Dei shows various degrees of similarity with glucosyltransferases and glucan-binding protein and contains A and C repeating units probably involved in glucan binding. Southern hybridization results showed that the dei probe from UAB108 hybridized to the same-size fragment in S. sobrinus (serotype d and g) DNA, to a different-size fragment in S. downei (serotype h) and S. cricetus (serotype a), and not at all to DNAs from other mutans group of streptococci.     

Mycobiota associated with insect galleries in walnut with thousand cankers disease reveals a potential natural enemy against Geosmithia morbida

Thousand Cankers Disease (TCD) affects Juglans and Pterocarya species. This disease poses not only a major threat to the nut and timber industries but also to native stands of walnut trees. Galleries created by Pityophthorus juglandis (vector) are colonized by the fungus Geosmithia morbida (causal agent of necrosis). It is unknown if other fungi colonizing these galleries might act antagonistically towards G. morbida. The objectives of this study were to: (1) characterize the fungal community associated with TCD-infected trees and (2) develop a pilot study addressing their potential antagonism towards G. morbida. We collected non-Geosmithia fungi from ten TCD-infected walnut trees from California and Tennessee. Four hundred and fifty-seven isolates, representing sixty-five Operational Taxonomic Units (99 % ITS similarity) were obtained. Fungal communities were found to be highly diverse. Ophiostoma dominated the communities associated with TCD-compromised trees from California, whereas Trichoderma dominated TCD-compromised trees in Tennessee. Six Trichoderma isolates showed varying levels of antagonism against three isolates of G. morbida, suggesting potential applications for the biological control of TCD. Furthermore, results from this study contribute to the growing knowledge about the observed differential disease development between the western and eastern USA and could overall impact our understanding of TCD etiology.     

Phosphatidylinositol-4-Phosphate 5-Kinases and Phosphatidylinositol 4,5-Bisphosphate Synthesis in the Brain

The predominant pathway for phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P₂) synthesis is thought to be phosphorylation of phosphatidylinositol 4-phosphate at the 5 position of the inositol ring by type I phosphatidylinositol phosphate kinases (PIPK): PIPKIα, PIPKIβ, and PIPKIγ. PIPKIγ has been shown to play a role in PI(4,5)P₂ synthesis in brain, and the absence of PIPKIγ is incompatible with postnatal life. Conversely, mice lacking PIPKIα or PIPKIβ (isoforms are referred to according to the nomenclature of human PIPKIs) live to adulthood, although functional effects in specific cell types are observed. To determine the contribution of PIPKIα and PIPKIβ to PI(4,5)P₂ synthesis in brain, we investigated the impact of disrupting multiple PIPKI genes. Our results show that a single allele of PIPKIγ, in the absence of both PIPKIα and PIPKIβ, can support life to adulthood. In addition, PIPKIα alone, but not PIPKIβ alone, can support prenatal development, indicating an essential and partially overlapping function of PIPKIα and PIPKIγ during embryogenesis. This is consistent with early embryonic expression of PIPKIα and PIPKIγ but not of PIPKIβ. PIPKIβ expression in brain correlates with neuronal differentiation. The absence of PIPKIβ does not impact embryonic development in the PIPKIγ knock-out (KO) background but worsens the early postnatal phenotype of the PIPKIγ KO (death occurs within minutes rather than hours). Analysis of PIP₂ in brain reveals that only the absence of PIPKIγ significantly impacts its levels. Collectively, our results provide new evidence for the dominant importance of PIPKIγ in mammals and imply that PIPKIα and PIPKIβ function in the generation of specific PI(4,5)P₂ pools that, at least in brain, do not have a major impact on overall PI(4,5)P₂ levels.