Herbivore selection of plant hosts and plant responses to insect colonization have been subjects of intense investigations. A growing body of evidence suggests that, for successful colonization to occur, (effector/virulence) proteins in insect saliva must modulate plant defense responses to the benefit of the insect. A range of insect saliva proteins that modulate plant defense responses have been identified, but there is no direct evidence that these proteins are delivered into specific plant tissues and enter plant cells. Aphids and other sap-sucking insects of the order Hemiptera use their specialized mouthparts (stylets) to probe plant mesophyll cells until they reach the phloem cells for long-term feeding. Here, we show, by immunogold-labeling of ultrathin sections of aphid feeding sites, that an immuno-suppressive aphid effector localizes in the cytoplasm of mesophyll cells near aphid stylets but not in cells further away from aphid feeding sites. In contrast, another aphid effector protein localizes in the sheaths composed of gelling saliva that surround the aphid stylets. Thus, insects deliver effectors directly into plant tissue. Moreover, different aphid effectors locate extracellularly in the sheath saliva or are introduced into the cytoplasm of plant cells. [Formula: see text] Copyright © 2016 The Author(s). This is an open-access article distributed under the CC BY-NC-ND 4.0 International license .

Starch degradation in barley endosperm provides carbon for early seedling growth, but the control of this process is poorly understood. We investigated whether endosperm cell wall degradation is an important determinant of the rate of starch degradation. We identified iminosugar inhibitors of enzymes that degrade the cell wall component arabinoxylan. The iminosugar 1,4-dideoxy-1, 4-imino-l-arabinitol (LAB) inhibits arabinoxylan arabinofuranohydrolase (AXAH) but does not inhibit the main starch-degrading enzymes α- and β-amylase and limit dextrinase. AXAH activity in the endosperm appears soon after the onset of germination and resides in dimers putatively containing two isoforms, AXAH1 and AXAH2. Upon grain imbibition, mobilisation of arabinoxylan and starch spreads across the endosperm from the aleurone towards the crease. The front of arabinoxylan degradation precedes that of starch degradation. Incubation of grains with LAB decreases the rate of loss of both arabinoxylan and starch, and retards the spread of both degradation processes across the endosperm. We propose that starch degradation in the endosperm is dependent on cell wall degradation, which permeabilises the walls and thus permits rapid diffusion of amylolytic enzymes. AXAH may be of particular importance in this respect. These results provide new insights into the mobilization of endosperm reserves to support early seedling growth.

Starch granule initiation is poorly understood at the molecular level. The glucosyltransferase, STARCH SYNTHASE 4 (SS4), plays a central role in granule initiation in Arabidopsis leaves, but its function in cereal endosperms is unknown. We investigated the role of SS4 in wheat, which has a distinct spatiotemporal pattern of granule initiation during grain development.(2) We generated TILLING mutants in tetraploid wheat (Triticum turgidum) that are defective in both SS4 homoeologs. The morphology of endosperm starch was examined in developing and mature grains.(3) SS4 deficiency led to severe alterations in endosperm starch granule morphology. During early grain development, while the wild type initiated single 'A-type' granules per amyloplast, most amyloplasts in the mutant formed compound granules due to multiple initiations. This phenotype was similar to mutants deficient in B-GRANULE CONTENT 1 (BGC1). SS4 deficiency also reduced starch content in leaves and pollen grains.(4) We propose that SS4 and BGC1 are required for the proper control of granule initiation during early grain development that leads to a single A-type granule per amyloplast. The absence of either protein results in a variable number of initiations per amyloplast and compound granule formation.

Post-translational thio-acylation of a fusion protein between the alpha2A-adrenoceptor and the alpha subunit of the G protein G(o1) is both dynamic and regulated by agonist binding. Incorporation of [3H]palmitate into the fusion protein was reduced substantially in the presence of the agonist adrenaline. This was dependent on the concentration of adrenaline and correlated with occupancy of the ligand binding site. Both the receptor and G-protein elements of the fusion construct incorporated [3H]palmitate but this occurred more rapidly for the G-protein element and regulation of acylation by the agonist occurred only for the G protein. The kinetics of de-palmitoylation of the alpha2A-adrenoceptor-Galpha(o1) fusion were accelerated markedly by agonist. Again, this reflected modulation of the G protein but not of the receptor. Agonist-induced regulation of the kinetics of thio-acylation of the G protein was abolished, however, in a mutant unable to bind guanosine 5'-[gamma-[35S]thio]triphosphate ([35S]GTP[S]) in response to adrenaline. Despite the dynamic nature of the post-translational acylation and its regulation by agonist, the ability of adrenaline to activate the G protein, monitored by stimulation of the binding of [35S]GTP[S] to such fusion constructs, was unaffected by the palmitoylation potential of either the receptor or G-protein element.

The spatial and temporal patterns by which starch granules initiate vary greatly between species and organs, but molecular factors that contribute to these diverse patterns are poorly understood. In wheat, the endosperm of grains contains two distinct types of starch granules, where the synthesis of large A-type granules is initiated during early grain development, followed by small B-type granules that initiate c. 10-15 days after the A-type granules, whereas the leaves contain uniformly-sized starch granules. Here, we reveal distinct organ-specific roles of the MYOSIN-RESEMBLING CHLOROPLAST PROTEIN (MRC) in regulating granule initiation in the endosperm and leaves of wheat. We isolated three independent TILLING mutants of tetraploid wheat (Triticum turgidum cv. Kronos) with premature stop or missense mutations in the A genome homeolog, which is the only active homeolog in tetraploid wheat due to a disruption of the B genome homeolog that occurred shortly after wheat hybridisation. The mutants had significantly smaller A-type granules and a higher relative volume of B-type granules than the wild type. Quantification of starch granule size distributions and microscopy during grain development showed that B-type granules initiated 15 - 20 dpa in the wild-type, but as early as 10 dpa in the mrc-1 mutant, suggesting a novel role of MRC in suppressing B-type granule initiation during early grain development. By contrast, MRC has a role in promoting granule initiation in leaves, as the mutants carrying premature stop mutations in MRC had fewer granules per chloroplast in comparison to the wild type. Overall, these contrasting roles of MRC among wheat organs provide new insight into functional diversification of granule initiation proteins to facilitate the diverse patterns of granule initiation observed across species and organs.

Herbivore selection of plant hosts and plant responses to insect colonization have been subjects of intense investigations. A growing body of evidence suggests that for successful colonization to occur, (effector/virulence) proteins in insect saliva must modulate plant defense responses to the benefit of the insect. A range of insect saliva proteins that modulate plant defense responses have been identified, but there is no direct evidence that these proteins are delivered into specific plant tissues and enter plant cells. Aphids and other sap-sucking insects of the order Hemiptera use their specialized mouthparts (stylets) to probe plant mesophyll cells, until they reach the . CC-BY-NC4.0 International license not peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was . http://dx.doi.org/10.1101/071811 doi: bioRxiv preprint first posted online Aug. 30, 2016; Mugford. MPMI. 2 phloem cells for long-term feeding. Here we show by immunogold-labeling of ultrathin sections of aphid feeding sites that an immuno-suppressive aphid effector localizes in the cytoplasm of mesophyll cells near aphid stylets, but not in cells further away from aphid feeding sites. In contrast, another aphid effector protein localizes in the sheaths composed of gelling saliva that surround the aphid stylets. Thus, insects deliver effectors directly into plant tissue. Moreover, different aphid effectors locate extracellularly in the sheath saliva or are introduced into the cytoplasm of plant cells.Introduction:

Ammonia-oxidising archaea (AOA) are environmentally important microorganisms involved in the biogeochemical cycling of nitrogen. Routine cultivation of AOA is exclusively performed in liquid cultures and reports on their growth on solid medium are scarce. The ability to grow AOA on solid medium would be beneficial for not only the purification of enrichment cultures but also in developing of genetic tools. The aim of this study was to develop a reliable method for growing individual colonies from AOA cultures on solid medium. Three phylogenetically distinct AOA strains were tested: ‘Candidatus Nitrosocosmicus franklandus C13’, Nitrososphaera viennensis EN76 and ‘Candidatus Nitrosotalea sinensis Nd2’. Of the gelling agents tested, agar and Bacto-agar severely inhibited growth of all three strains. In contrast, both ‘Ca. N. franklandus C13’ and N. viennensis EN76 tolerated Phytagel™ while the acidophilic ‘Ca. N. sinensis Nd2’ was completely inhibited. Based on these observations, we developed a Liquid-Solid (LS) method that involves immobilising cells in Phytagel™ and overlaying with liquid medium. This approach resulted in the development of visible distinct colonies from ‘Ca. N. franklandus C13’ and N. viennensis EN76 cultures and lays the groundwork for the genetic manipulation of this group of microorganisms.

Fungi have evolved an array of spore discharge and dispersal processes. Here, we developed a theoretical model that explains the ejection mechanics of aeciospore liberation in the stem rust pathogen Puccinia graminis. Aeciospores are released from cluster cups formed on its Berberis host, spreading early-season inoculum into neighboring small-grain crops. Our model illustrates that during dew or rainfall, changes in aeciospore turgidity exerts substantial force on neighboring aeciospores in cluster cups whilst gaps between spores become perfused with water. This perfusion coats aeciospores with a lubrication film that facilitates expulsion, with single aeciospores reaching speeds of 0.053 to 0.754 m·s−1. We also used aeciospore source strength estimates to simulate the aeciospore dispersal gradient and incorporated this into a publicly available web interface. This aids farmers and legislators to assess current local risk of dispersal and facilitates development of sophisticated epidemiological models to potentially curtail stem rust epidemics originating on Berberis.