Morphological and developmental analysis of peripheral antennal chemosensory sensilla and central olfactory glomeruli in worker castes of Camponotus compressus (Fabricius, 1787)

The antennal lobes of different castes of the ant species Camponotus compressus show a marked diversity in the organization of their olfactory glomeruli. Notably, there is a significant difference in the number and size of glomeruli between the reproductives and the workers and among the different worker castes. In this report, we investigate the notion that these caste-specific differences in glomerular number might be accounted for, at least in part, by the differences in numbers of olfactory sensilla that target the antennal lobe. For this, we examine the number of sensilla on the antennal flagella of all the individual castes of C. compressus. This analysis reveals a striking correlation between sensillar number and the number of antennal glomeruli in a given caste. As a first step in investigating the causal mechanisms that might give raise to this correlation, we carry out an initial characterization of olfactory system development in the minor workers of C. compressus. We analyze the temporal pattern of innervations of the developing antennal lobe by olfactory sensory neuron axons. We document the development of the olfactory glomeruli in the antennal lobe during this process, which occurs during early pupal stages. Our findings provide the basis for future manipulative developmental studies on the role of sensory afferent number in glomerular development of different castes within the same species.     

Structure-based optimization of morpholino-triazines as PI3K and mTOR inhibitors

A virtual screen of our in-house database using various fingerprint techniques returned several triazine hits which were found to be mTOR inhibitors with a slight selectivity over PI3Kα. Using structure-guided lead optimization the inhibitory activity towards mTOR and PI3Kα was increased to the low nanomolar range. Exploiting shape differences in the binding-site allowed for the design of mTOR selective inhibitors. Focus on ligand efficiency ensured the inhibitors retained a low molecular weight and desirable drug-like properties.     

NTRC and chloroplast-generated reactive oxygen species regulate Pseudomonas syringae pv. tomato disease development in tomato and arabidopsis

Coronatine (COR)-producing pathovars of Pseudomonas syringae, including pvs. tomato, maculicola, and glycinea, cause important diseases on tomato, crucifers, and soybean, respectively, and produce symptoms with necrotic lesions surrounded by chlorosis. The chlorosis is mainly attributed to COR. However, the significance of COR-induced chlorosis in localized lesion development and the molecular basis of disease-associated cell death is largely unknown. To identify host (chloroplast) genes that play a role in COR-mediated chlorosis, we used a forward genetics approach using Nicotiana benthamiana and virus-induced gene silencing and identified a gene which encodes 2-Cys peroxiredoxin (Prxs) that, when silenced, produced a spreading hypersensitive or necrosis-like phenotype instead of chlorosis after COR application in a COI1-dependent manner. Loss-of-function analysis of Prx and NADPH-dependent thioredoxin reductase C (NTRC), the central players of a chloroplast redox detoxification system, resulted in spreading accelerated P. syringae pv. tomato DC3000 disease-associated cell death with enhanced reactive oxygen species (ROS) accumulation in a COR-dependent manner in tomato and Arabidopsis. Consistent with these results, virulent strain DC3000 suppressed the expression of Prx and NTRC in Arabidopsis and tomato during pathogenesis. However, interestingly, authentic COR suppressed the expression of Prx and NTRC in tomato but not in Arabidopsis, suggesting that COR in conjunction with other effectors may modulate ROS and cell death in different host species. Taken together, these results indicated that NTRC or Prx function as a negative regulator of pathogen-induced cell death in the healthy tissues that surround the lesions, and COR-induced chloroplast-localized ROS play a role in enhancing the disease-associated cell death.     

A GRAS-Type Transcription Factor with a Specific Function in Mycorrhizal Signaling

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Agrobacterium May Delay Plant Nonhomologous End-Joining DNA Repair via XRCC4 to Favor T-DNA Integration

Agrobacterium tumefaciens is a soilborne pathogen that causes crown gall disease in many dicotyledonous plants by transfer of a portion of its tumor-inducing plasmid (T-DNA) into the plant genome. Several plant factors that play a role in Agrobacterium attachment to plant cells and transport of T-DNA to the nucleus have been identified, but the T-DNA integration step during transformation is poorly understood and has been proposed to occur via nonhomologous end-joining (NHEJ)–mediated double-strand DNA break (DSB) repair. Here, we report a negative role of X-RAY CROSS COMPLEMENTATION GROUP4 (XRCC4), one of the key proteins required for NHEJ, in Agrobacterium T-DNA integration. Downregulation of XRCC4 in Arabidopsis and Nicotiana benthamiana increased stable transformation due to increased T-DNA integration. Overexpression of XRCC4 in Arabidopsis decreased stable transformation due to decreased T-DNA integration. Interestingly, XRCC4 directly interacted with Agrobacterium protein VirE2 in a yeast two-hybrid system and in planta. VirE2-expressing Arabidopsis plants were more susceptible to the DNA damaging chemical bleomycin and showed increased stable transformation. We hypothesize that VirE2 titrates or excludes active XRCC4 protein available for DSB repair, thus delaying the closure of DSBs in the chromosome, providing greater opportunity for T-DNA to integrate.     

Mutations in Arabidopsis Fatty Acid Amide Hydrolase Reveal That Catalytic Activity Influences Growth but Not Sensitivity to Abscisic Acid or Pathogens

Fatty acid amide hydrolase (FAAH) terminates the endocannabinoid signaling pathway that regulates numerous neurobehavioral processes in animals by hydrolyzing N-acylethanolamines (NAEs). Recently, an Arabidopsis FAAH homologue (AtFAAH) was identified, and several studies, especially those using AtFAAH overexpressing and knock-out lines, have suggested an in vivo role for FAAH in the catabolism of NAEs in plants. We previously reported that overexpression of AtFAAH in Arabidopsis resulted in accelerated seedling growth, and in seedlings that were insensitive to exogenous NAEs but hypersensitive to abscisic acid (ABA) and hypersusceptible to nonhost pathogens. Here we show that whereas the enhanced growth and NAE tolerance of the AtFAAH overexpressing seedlings depend on the catalytic activity of AtFAAH, hypersensitivity to ABA and hypersusceptibility to nonhost pathogens are independent of its enzymatic activity. Five amino acids known to be critical for rat FAAH activity are also conserved in AtFAAH (Lys-205, Ser-281, Ser-282, Ser-305, and Arg-307). Site-directed mutation of each of these conserved residues in AtFAAH abolished its hydrolytic activity when expressed in Escherichia coli, supporting a common catalytic mechanism in animal and plant FAAH enzymes. Overexpression of these inactive AtFAAH mutants in Arabidopsis showed no growth enhancement and no NAE tolerance, but still rendered the seedlings hypersensitive to ABA and hypersusceptible to nonhost pathogens to a degree similar to the overexpression of the native AtFAAH. Taken together, our findings suggest that the AtFAAH influences plant growth and interacts with ABA signaling and plant defense through distinctly different mechanisms.     

Sulfonate protecting groups. Improved synthesis of scyllo-inositol and its orthoformate from myo-inositol

A convenient high yielding method for the preparation of scyllo-inositol and its orthoformate from myo-inositol, without involving chromatography is described. myo-Inositol 1,3,5-orthoformate was benzoylated to obtain 2-O-benzoyl-myo-inositol 1,3,5-orthoformate. This diol was tosylated and the benzoyl group removed by aminolysis in a one-pot procedure to obtain 4,6-di-O-tosyl-myo-inositol 1,3,5-orthoformate. Swern oxidation of the ditosylate, followed by sodium borohydride reduction and methanolysis of tosylates gave scyllo-inositol 1,3,5-orthoformate (isolated as the triacetate). Aminolysis of the acetates followed by acid hydrolysis of the orthoformate moiety with trifluoroacetic acid gave scyllo-inositol in an overall yield of 64%.     

Expression of the Arabidopsis histone H2A-1 gene correlates with susceptibility to Agrobacterium transformation

Transformation of plant cells by Agrobacterium tumefaciens involves both bacterial virulence proteins and host proteins. We have previously shown that the Arabidopsis thaliana gene H2A-1 (RAT5), which encodes histone H2A-1, is involved in T-DNA integration into the plant genome. Mutation of RAT5 results in a severely decreased frequency of transformation, whereas overexpression of RAT5 enhances the transformation frequency (Mysore et al., 2000b). We show here that the expression pattern of RAT5 correlates with plant root cells most susceptible to transformation. As opposed to a cyclin-GUS fusion gene whose expression is limited to meristematic tissues, the H2A-1 gene is expressed in many non-dividing cells. Under normal circumstances, the H2A-1 gene is expressed in the elongation zone of the root, the region that is most susceptible to Agrobacterium transformation. In addition, when roots are incubated on medium containing phytohormones, a concomitant shift in H2A-1 expression and transformation susceptibility to the root base is observed. Inoculation of root segments with a transfer-competent, but not a transformation-deficient Agrobacterium strain induces H2A-1 expression. Furthermore, pre-treatment of Arabidopsis root segments with phytohormones both induces H2A-1 expression and increases the frequency of Agrobacterium transformation. Our results suggest that the expression of the H2A-1 gene is both a marker for, and a predictor of, plant cells most susceptible to Agrobacterium transformation.