Kinase-dead mutation: A novel strategy for improving soybean resistance to soybean cyst nematode Heterodera glycines

Protein kinases phosphorylate proteins for functional changes and are involved in nearly all cellular processes, thereby regulating almost all aspects of plant growth and development, and responses to biotic and abiotic stresses. We generated two independent co-expression networks of soybean genes using control and stress response gene expression data and identified 392 differentially highly interconnected kinase hub genes among the two networks. Of these 392 kinases, 90 genes were identified as “syncytium highly connected hubs”, potentially essential for activating kinase signalling pathways in the nematode feeding site. Overexpression of wild-type coding sequences of five syncytium highly connected kinase hub genes using transgenic soybean hairy roots enhanced plant susceptibility to soybean cyst nematode (SCN; Heterodera glycines) Hg Type 0 (race 3). In contrast, overexpression of kinase-dead variants of these five syncytium kinase hub genes significantly enhanced soybean resistance to SCN. Additionally, three of the five tested kinase hub genes enhanced soybean resistance to SCN Hg Type 1.2.5.7 (race 2), highlighting the potential of the kinase-dead approach to generate effective and durable resistance against a wide range of SCN Hg types. Subcellular localization analysis revealed that kinase-dead mutations do not alter protein cellular localization, confirming the structure–function of the kinase-inactive variants in producing loss-of-function phenotypes causing significant decrease in nematode susceptibility. Because many protein kinases are highly conserved and are involved in plant responses to various biotic and abiotic stresses, our approach of identifying kinase hub genes and their inactivation using kinase-dead mutation could be translated for biotic and abiotic stress tolerance.     

Enemy at the Gates: Interaction-Specific Stomatal Responses to Pathogenic Challenge

Stomata regulate gas exchange and their closure in response to pathogens may, in some cases, contribute to resistance. However, in the cereal mildew and rust systems, stomatal closure follows establishment of compatible infections. In incompatible systems, expression of major (R) gene controlled hypersensitive responses (HR), causes drastic, permanent stomatal dysfunction: stomata become locked open following powdery mildew attack and locked shut following rust attack. Thus, stomatal locking can be a hitherto unsuspected negative consequence of R gene resistance that carries a physiological cost affecting plant performance.Key Words: stomata, rust, mildew, hypersensitive response, stomatal lock-up     

The high-resolution NMR structure of the R21A Spc-SH3:P41 complex: Understanding the determinants of binding affinity by comparison with Abl-SH3

Background  

SH3 domains are small protein modules of 60–85 amino acids that bind to short proline-rich sequences with moderate-to-low affinity and specificity. Interactions with SH3 domains play a crucial role in regulation of many cellular processes (some are related to cancer and AIDS) and have thus been interesting targets in drug design. The decapeptide APSYSPPPPP (p41) binds with relatively high affinity to the SH3 domain of the Abl tyrosine kinase (Abl-SH3), while it has a 100 times lower affinity for the α-spectrin SH3 domain (Spc-SH3).     

Internodal myelination during development quantitated using X-ray diffraction

Characterizing the formation, accretion, and stability of myelin during development, maturation, and senescence is important for better understanding critical periods in the function of the nervous system in normal growth and following environmental insult or genetic mutation. Although there are numerous studies on the ultrastructural, biochemical, and genetic aspects of myelin development and maturation, few have used X-ray diffraction (XRD), which can rapidly provide unique metrics about internodal myelin based on measurements from whole, unfixed tissue. Besides periodicity (the classic attribute of internodal myelin measured by XRD), other parameters include: relative amount of myelin, membrane dimensions, and packing disorder. To provide a baseline for future experiments on myelin structural integrity, we used XRD to characterize internodal myelin as a function of age (from 5 to 495 days) in the mouse, a species increasingly used for developing transgenic models of human neurological diseases. As expected, the relative amount of myelin increased with age in both PNS and CNS, with the most rapid accumulation occurring in the youngest age group. Changes in rate of myelin accretion yielded three distinct age brackets during which small but significant changes in structural parameters were detected: in PNS, myelin period increased, packing distortion decreased, width of extracellular apposition (EXT) decreased, and widths of cytoplasmic apposition (CYT) and lipid bilayer (LPG) increased; in CNS, myelin period decreased, packing distortion decreased, EXT and CYT decreased, and LPG increased. We propose that the data obtained here can serve as a basis for rapidly detecting abnormal pathologies during myelination.     

Species delimitation of the liquorice tribe (Leguminosae: Glycyrrhizeae) based on phylogenomic and machine learning analyses

The liquorice tribe Glycyrrhizeae is a leguminous herbaceous group of plants comprised of the genera Glycyrrhiza and Glycyrrhizopsis. Some Glycyrrhiza taxa contain glycyrrhizin, a pharmacologically significant sweet substance that also has applications in crafting industrial materials. Here, we utilized an expanded taxon sampling of Glycyrrhizeae to reconstruct the phylogenetic relationships in the tribe based on genome skimming data, including whole chloroplast genomes, nuclear ribosomal DNA, and low-copy nuclear DNA. We also launched machine learning analysis (MLA) for one species pair with controversial taxonomic boundary. The integrated results indicated Glycyrrhizopsis should be split from Glycyrrhiza, while the former genus Meristotropis should be treated as part of Glycyrrhiza. Glycyrrhizopsis includes two species, Glycyrrhizopsis asymmetrica and Glycyrrhizopsis flavescens, and we recognize 13 species in Glycyrrhiza: Glycyrrhiza acanthocarpa, Glycyrrhiza astragalina, Glycyrrhiza bucharica, Glycyrrhiza echinata, Glycyrrhiza foetida, Glycyrrhiza glabra, Glycyrrhiza gontscharovii, Glycyrrhiza lepidota, Glycyrrhiza macedonica, Glycyrrhiza pallidiflora, Glycyrrhiza squamulosa, Glycyrrhiza triphylla, and Glycyrrhiza yunnanensis. We propose a broader G. glabra that includes former Glycyrrhiza aspera, G. glabra s.s., Glycyrrhiza inflata, and Glycyrrhiza uralensis, and represents the glycyrrhizin-contained medicinal group. Our ancestral state inferences show the ancestor of Glycyrrhiza lacked glycyrrhizin, and the presence of glycyrrhizin evolved twice within Glycyrrhiza during the last one million years. Our integrative phylogenomics-MLA study not only provides new insights into long-standing taxonomic controversies of Glycyrrhizeae, but also represents a useful approach for future taxonomic studies on other plant taxa.     

Rearrangements of integral membrane components during in vitro aging of sheep erythrocyte membranes

In vitro aged sheep erythrocytes and sheep erythrocyte ghosts spontaneously release vesicles that consist of long protrusions affixed to flattened headlike structures. The intramembranous particles seen on the protoplasmic face of freeze fracture electron micrographs of vesicle protrusions are arranged in paired particle rows. On the equivalent fracture face of headlike structures, the particle density is low; if particles are present, they are clustered along the rim of the flattened headlike structure and at the junction with the protrusion. The released vesicles are depleted of the intramembranous particles seen on the exoplasmic face of ghost but retain almost exclusively particles of the protoplasmic face. Correspondingly, the exoplasmic face of ghosts that have released vesicles reveals a 28 percent higher density of intramembranous particles than that of fresh ghosts. Purified vesicles are depleted of spectrin but retain integral membrane proteins, with one of an apparent mol wt of 160,000 accounting for nearly 50 percent of the total protein (Lutz, H.U.,R. Barber, and R.F. McGuire. 1976. J. Biol. Chem. 251:3500-3510). When vesicles are modified with the cleavable cross-linking reagent [(35)S]dithiobis (succinimidyl propionate)at 0 degrees C, the 160,000 mol wt protein is rapidly converted to disulfide-linked dimers and higher oligomers. Exposure of intact ghosts to the reagent in the same way fails to yield equivalent polymers. A comparison of the morphological and biochemical aspects of ghosts and vesicles suggest that a marked rearrangement of membrane proteins accompanies the supramolecular redistribution of intramembranous particles during spontaneous vesiculation. The results also suggest that the paired particles of the protoplasmic face of vesicle protrusions are arranged in paired helices and contain the 160,000 mol wt protein as dimers.     

Investigations on the detrimental effect of prostaglandin F2α-regulated estrus on the number and condition of sperm in the reproductive tract of the ewe

Ewes in the luteal phase of the estrous cycle were treated with prostaglandin F₂α (PGF), mated to rams at the ensuing estrus 2 days later, and necropsied at 2 or 23 hr after mating. At 2 hr after mating, ewes in PGF-regulated estrus had significantly fewer sperm in the middle and anterior one-thirds of the cervix and in the uterus than did ewes mated during natural estrus. At 23 hr, soon after ovulation, significantly fewer ewes in PGF-regulated estrus had sperm in the oviducts than did ewes in natural estrus.In Experiment 2, ewes in PGF-regulated or natural estrus were laparotomized, inseminated by deposition of semen in the uterine lumen, and necropsied 2 or 23 hr later. Intrauterine insemination prevented most of the reduction in sperm numbers in the reproductive tract at PGF-regulated estrus.In Experiment 3, ewes in PGF-regulated or natural estrus were either mated to rams or inseminated in the uterine lumen and necropsied 2 hr later. Sperm were recovered from three segments of the cervix and were counted and evaluated for motility, response to live-dead staining, and acrosomal morphology. Intrauterine insemination again reduced the detrimental effect of PGF-regulated estrus on sperm numbers. However, the percentages of sperm recovered from the cervix that were motile, live, and had normal acrosomes were much lower in ewes in PGF-regulated estrus than in ewes in natural estrus. Compared with natural mating, intrauterine insemination reduced but did not eliminate the detrimental effects of PGF-regulated estrus on the viability and morphology of sperm. Regulating estrus with PGF resulted in damage to sperm in the cervix regardless of whether sperm reached the cervix from the vagina or from the uterus.