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).     

Characterization of the Fungal Gibberellin Desaturase as a 2-Oxoglutarate-Dependent Dioxygenase and Its Utilization for Enhancing Plant Growth

The biosynthesis of gibberellic acid (GA₃) by the fungus Fusarium fujikuroi is catalyzed by seven enzymes encoded in a gene cluster. While four of these enzymes are characterized as cytochrome P450 monooxygenases, the nature of a fifth oxidase, GA₄ desaturase (DES), is unknown. DES converts GA₄ to GA₇ by the formation of a carbon-1,2 double bond in the penultimate step of the pathway. Here, we show by expression of the des complementary DNA in Escherichia coli that DES has the characteristics of a 2-oxoglutarate-dependent dioxygenase. Although it has low amino acid sequence homology with known 2-oxoglutarate-dependent dioxygenases, putative iron- and 2-oxoglutarate-binding residues, typical of such enzymes, are apparent in its primary sequence. A survey of sequence databases revealed that homologs of DES are widespread in the ascomycetes, although in most cases the homologs must participate in non-gibberellin (GA) pathways. Expression of des from the cauliflower mosaic virus 35S promoter in the plant species Solanum nigrum, Solanum dulcamara, and Nicotiana sylvestris resulted in substantial growth stimulation, with a 3-fold increase in height in S. dulcamara compared with controls. In S. nigrum, the height increase was accompanied by a 20-fold higher concentration of GA₃ in the growing shoots than in controls, although GA₁ content was reduced. Expression of des was also shown to partially restore growth in plants dwarfed by ectopic expression of a GA 2-oxidase (GA-deactivating) gene, consistent with GA₃ being protected from 2-oxidation. Thus, des has the potential to enable substantial growth increases, with practical implications, for example, in biomass production.The GAs are a class of diterpenoid hormones that regulate many aspects of growth and development in plants, including stem extension (Thomas and Hedden, 2006). Despite being ubiquitous in higher plants, they were first discovered as secondary metabolites of the plant pathogenic fungus Gibberella fujikuroi, the causative agent of the bakanae disease of rice (Oryza sativa; Phinney, 1983). This fungus is now known to comprise a group of reproductively isolated species or mating populations, the rice pathogen belonging to mating group C and assigned the name Fusarium fujikuroi (Leslie and Summerell, 2006; Kvas et al., 2009). Details of the GA biosynthetic pathways in both plants and the fungus are known in considerable detail and have revealed that, although they give rise to common metabolites, the pathways utilize different types of enzymes for several steps and appear to have evolved independently (Hedden et al., 2001; Bömke and Tudzynski, 2009).Higher plants differ from the GA-producing fungi by possessing the means for GA inactivation, which is necessary to allow precise regulation of their GA concentration. In contrast, the fungi are not dependent on GAs for their development but produce and secrete large quantities of the compounds to modify the behavior of their hosts. It has been shown that GAs interfere with plant defense by suppressing jasmonate signaling and may thus compromise the host’s ability to evade fungal infection (Navarro et al., 2008; Hou et al., 2010). An apparent ubiquitous inactivation mechanism involves 2β-hydroxylation (Thomas et al., 1999), the effect of which reduces binding of the GA within the active site of the GID1 receptor (Murase et al., 2008). However, GAs such as GA₃ and GA₅, which are unsaturated on C-2, are protected from 2β-hydroxylation and, as a consequence, would be expected to be turned over more slowly than their saturated analogs (King et al., 2008). In accordance with the requirement to regulate GA content, shoots of higher plants contain relatively little 1,2-unsaturated GAs, although developing seeds of some species contain substantial quantities. They are produced in a two-step reaction via a 2,3-dehydro intermediate, which is then hydroxylated on C-3β with rearrangement of the double bond from C-2,3 to C-1,2 (Albone et al., 1990). The reactions are catalyzed by GA 3-oxidase-type enzymes, with a single enzyme catalyzing both reactions in cereal shoots to produce GA₃ from GA₂₀ as a minor by-product of GA₁ biosynthesis (Itoh et al., 2001; Appleford et al., 2006; Fig. 1). In developing seeds of Marah macrocarpus, which contain high concentrations of the 1,2-unsaturated GA, GA₇, the formation of this GA from GA₉ requires the activities of two functionally different GA 3-oxidases acting sequentially (Ward et al., 2010). However, direct formation of GA₇ from GA₄, such as occurs in F. fujikuroi, is not usual in higher plants.Open in a separate windowFigure 1.The GA biosynthetic pathway in plants and F. fujikuroi. The fungal pathway to GA₃ is indicated by the thick gray arrow. DES catalyzes the conversion of GA₄ to GA₇.While the late stages of GA biosynthesis in higher plants, including desaturation when it occurs and 2β-hydroxylation, are catalyzed by 2-oxoglutarate-dependent dioxygenases (ODDs), these enzymes have not been shown to be involved in GA biosynthesis in fungi. F. fujikuroi contains a cluster of seven genes for GA biosynthesis, including a geranylgeranyl diphosphate synthase that is specific to the GA pathway and a bifunctional terpene cyclase that converts geranylgeranyl diphosphate to ent-kaurene in two steps via ent-copalyl diphosphate (for review, see Hedden et al. [2001]; Bömke and Tudzynski [2009]). The formation of GA₃ from ent-kaurene requires the activity of five oxidases (Fig. 1), four of which are cytochrome P450 monooxygenases: P450-4 (ent-kaurene oxidase) oxidizes ent-kaurene to ent-kaurenoic acid (Tudzynski et al., 2001), which is converted to GA₁₄ by P450-1 (GA₁₄ synthase; Rojas et al., 2001); P450-2 functions as a GA 20-oxidase, converting GA₁₄ to GA₄ (Tudzynski et al., 2002), while, in the final step of the pathway, P450-3 13-hydroxylates GA₇ to form GA₁₃ (Tudzynski et al., 2003). However, the nature of the desaturase (DES), which converts GA₄ to GA₇ (Fig. 1), is unknown. When first described, it was found to have closest, albeit weak, homology to a component of the 7α-cephem-methoxylase from Nocardia lactamdurans, giving little indication of its mechanism (Tudzynski et al., 2003). Besides F. fujikuroi, several other ascomycetes, including Sphaceloma manihoticola (Bömke et al., 2008), Phaeosphaeria spp. (Kawaide, 2006), and two other species of the G. fujikuroi species complex, Fusarium konzum (Malonek et al., 2005) and Fusarium sacchari (Troncoso et al., 2010), have been shown to synthesize GAs, although the first two species do not carry out the desaturation step and do not contain a desaturase gene.The promotion of vegetative growth offers potential benefits, for example, in biomass production (Demura and Ye, 2010). In order to test the hypothesis that growth could be stimulated by increasing the shoot concentrations of GAs that are unsaturated on C-2 and therefore resistant to 2β-hydroxylation, we introduced the fungal desaturase gene into plants. The feasibility of this approach was reinforced by the demonstration that DES has the characteristics of an ODD and, therefore, would be expected to function in higher plants.     

Conditional embryonic lethality to improve the sterile insect technique in Ceratitis capitata (Diptera: Tephritidae)

Background  

The sterile insect technique (SIT) is an environment-friendly method used in area-wide pest management of the Mediterranean fruit fly Ceratitis capitata (Wiedemann; Diptera: Tephritidae). Ionizing radiation used to generate reproductive sterility in the mass-reared populations before release leads to reduction of competitiveness.     

Syndromic Management and STI Control in Urban Peru

Background

Syndromic management is an inexpensive and effective method for the treatment of symptomatic sexually transmitted infections (STIs), but its effectiveness as a method of STI control in at-risk populations is questionable. We sought to determine the potential utility of syndromic management as a public health strategy to control STI transmission in high-risk populations in urban Peru.

Methodology

We surveyed 3,285 at-risk men and women from three Peruvian cities from 2003–05. Participants were asked about the presence of genital ulcers, discharge, or dysuria in the preceding six months. Participants reporting symptoms were asked about subsequent health-seeking and partner notification behavior. Urine and vaginal swab samples were tested for Neisseria gonorrhoeae and Chlamydia trachomatis by nucleic acid testing. Serum was tested for syphilis and Herpes Simplex Virus-Type 2 antibodies.

Findings

Recent urogenital discharge or dysuria was reported by 42.1% of participants with gonorrhea or chlamydia versus 28.3% of participants without infection. Genital ulceration was reported by 6.2% of participants with, and 7.4% of participants without, recent syphilis. Many participants reporting symptoms continued sexual activity while symptomatic, and approximately half of all symptomatic participants sought treatment. The positive and negative predictive values of urogenital discharge or genital ulcer disease in detecting STIs that are common in the study population were 14.4% and 81.5% for chlamydia in women and 8.3% and 89.5% for syphilis among gay-identified men.

Conclusions

In our study, STIs among high-risk men and women in urban Peru were frequently asymptomatic and symptomatic participants often remained sexually active without seeking treatment. Additional research is needed to assess the costs and benefits of targeted, laboratory-based STI screening as part of a comprehensive STI control program in developing countries.     

Molecular modeling and dynamics studies of purine nucleoside phosphorylase from Bacteroides fragilis

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of N-ribosidic bonds of purine nucleosides and deoxynucleosides, except adenosine, to generate ribose 1-phosphate and the purine base. This work describes for the first time a structural model of PNP from Bacteroides fragilis (Bf). We modeled the complexes of BfPNP with six different ligands in order to determine the structural basis for specificity of these ligands against BfPNP. Comparative analysis of the model of BfPNP and the structure of HsPNP allowed identification of structural features responsible for differences in the computationally determined ligand affinities. The molecular dynamics (MD) simulation was assessed to evaluate the overall stability of the BfPNP model. The superposition of the final onto the initial minimized structure shows that there are no major conformational changes from the initial model, which is consistent with the relatively low root mean square deviation (RMSD). The results indicate that the structure of the model was stable during MD, and does not exhibit loosely structured loop regions or domain terminals.     

Crystal structure and molecular dynamics studies of human purine nucleoside phosphorylase complexed with 7-deazaguanine

In humans, purine nucleoside phosphorylase (HsPNP) is responsible for degradation of deoxyguanosine, and genetic deficiency of this enzyme leads to profound T-cell mediated immunosuppression. HsPNP is a target for inhibitor development aiming at T-cell immune response modulation. Here we report the crystal structure of HsPNP in complex with 7-deazaguanine (HsPNP:7DG) at 2.75 Å. Molecular dynamics simulations were employed to assess the structural features of HsPNP in both free form and in complex with 7DG. Our results show that some regions, responsible for entrance and exit of substrate, present a conformational variability, which is dissected by dynamics simulation analysis. Enzymatic assays were also carried out and revealed that 7-deazaguanine presents a lower inhibitory activity against HsPNP (K_i = 200 μM). The present structure may be employed in both structure-based design of PNP inhibitors and in development of specific empirical scoring functions.     

Optimal feeding strategy of the temperate herbivorous fish Aplodactylus punctatus: the effects of food availability on digestive and reproductive patterns

In this study we examined differences in feeding behevior of populations of the marine temperate herbivorous fish Aplodactylus punctatus, in three different localities off the Chilean coast, which differ qualitatively and quantitatively in food availability. We test whether food selection follows optimal foraging strategics, whether there is any modification of the fishes' digestive tracts in relation to their diets, and whether differences in diet quality affect the allocation of energy into reproduction and maintenance in these populations. Samples of this fish and of the understore algal assemblages were taken seasonally from May 1989 to February 1990. For each population we analyzed dietary composition, weight of the digestive tract and of the food content, the condition factor (K), and the gonadosomatic index (GSI). Our results showed that the diet observed in the three populations closely resembled the differences in macro-algal abundance and composition among the three localities studied. Local differences in diet quality were inversely related to the amount of food consumption and size of the digestive tract, suggesting that under differential conditions of food availability A. punctatus is able to compensate for variations in food quality through a flexibility in its digestive strategies. The nutritional status (K) of individuals, and their reproductive pattern (GSI) were directly related to diet quality. These results indicate that although A. punctatus is able to adjust its digestive processes to different algal food regimes, the digestive modifications observed in food-poor environments are not sufficient to compensate for the lack of food and allow fish to reach the nutritional status and reproductive output reached in a food-rich environment. This study represents the first natural experiment demonstrating a direct relationship among food availability, feeding patterns, digestive processes, and reproductive effort.     

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.