Nader Aryamanesh

SUBA4: the interactive data analysis centre for Arabidopsis subcellular protein locations

The SUBcellular location database for Arabidopsis proteins (SUBA4, http://suba.live) is a comprehensive collection of manually curated published data sets of large-scale subcellular proteomics, fluorescent protein visualization, protein-protein interaction (PPI) as well as subcellular targeting calls from 22 prediction programs. SUBA4 contains an additional 35 568 localizations totalling more than 60 000 experimental protein location claims as well as 37 new suborganellar localization categories. The experimental PPI data has been expanded to 26 327 PPI pairs including 856 PPI localizations from experimental fluorescent visualizations. The new SUBA4 user interface enables users to choose quickly from the filter categories: ‘subcellular location’, ‘protein properties’, ‘protein–protein interaction’ and ‘affiliations’ to build complex queries. This allows substantial expansion of search parameters into 80 annotation types comprising 1 150 204 new annotations to study metadata associated with subcellular localization. The ‘BLAST’ tab contains a sequence alignment tool to enable a sequence fragment from any species to find the closest match in Arabidopsis and retrieve data on subcellular location. Using the location consensus SUBAcon, the SUBA4 toolbox delivers three novel data services allowing interactive analysis of user data to provide relative compartmental protein abundances and proximity relationship analysis of PPI and coexpression partners from a submitted list of Arabidopsis gene identifiers.

Suppression of the auxin response pathway enhances susceptibility to Phytophthora cinnamomi while phosphite-mediated resistance stimulates the auxin signalling pathway

BackgroundPhytophthora cinnamomi is a devastating pathogen worldwide and phosphite (Phi), an analogue of phosphate (Pi) is highly effective in the control of this pathogen. Phi also interferes with Pi starvation responses (PSR), of which auxin signalling is an integral component. In the current study, the involvement of Pi and the auxin signalling pathways in host and Phi-mediated resistance to P. cinnamomi was investigated by screening the Arabidopsis thaliana ecotype Col-0 and several mutants defective in PSR and the auxin response pathway for their susceptibility to this pathogen. The response to Phi treatment was also studied by monitoring its effect on Pi- and the auxin response pathways.ResultsHere we demonstrate that phr1-1 (phosphate starvation response 1), a mutant defective in response to Pi starvation was highly susceptible to P. cinnamomi compared to the parental background Col-0. Furthermore, the analysis of the Arabidopsis tir1-1 (transport inhibitor response 1) mutant, deficient in the auxin-stimulated SCF (Skp1 − Cullin − F-Box) ubiquitination pathway was also highly susceptible to P. cinnamomi and the susceptibility of the mutants rpn10 and pbe1 further supported a role for the 26S proteasome in resistance to P. cinnamomi. The role of auxin was also supported by a significant (P < 0.001) increase in susceptibility of blue lupin (Lupinus angustifolius) to P. cinnamomi following treatment with the inhibitor of auxin transport, TIBA (2,3,5-triiodobenzoic acid). Given the apparent involvement of auxin and PSR signalling in the resistance to P. cinnamomi, the possible involvement of these pathways in Phi mediated resistance was also investigated. Phi (especially at high concentrations) attenuates the response of some Pi starvation inducible genes such as AT4, AtACP5 and AtPT2 in Pi starved plants. However, Phi enhanced the transcript levels of PHR1 and the auxin responsive genes (AUX1, AXR1and AXR2), suppressed the primary root elongation, and increased root hair formation in plants with sufficient Pi.ConclusionsThe auxin response pathway, particularly auxin sensitivity and transport, plays an important role in resistance to P. cinnamomi in Arabidopsis, and phosphite-mediated resistance may in some part be through its effect on the stimulation of the PSR and auxin response pathways.

Finding the Subcellular Location of Barley, Wheat, Rice and Maize Proteins: The Compendium of Crop Proteins with Annotated Locations (cropPAL)

Barley, wheat, rice and maize provide the bulk of human nutrition and have extensive industrial use as agricultural products. The genomes of these crops each contains >40,000 genes encoding proteins; however, the major genome databases for these species lack annotation information of protein subcellular location for >80% of these gene products. We address this gap, by constructing the compendium of crop protein subcellular locations called crop Proteins with Annotated Locations (cropPAL). Subcellular location is most commonly determined by fluorescent protein tagging of live cells or mass spectrometry detection in subcellular purifications, but can also be predicted from amino acid sequence or protein expression patterns. The cropPAL database collates 556 published studies, from >300 research institutes in >30 countries that have been previously published, as well as compiling eight pre-computed subcellular predictions for all Hordeum vulgare, Triticum aestivum, Oryza sativa and Zea mays protein sequences. The data collection including metadata for proteins and published studies can be accessed through a search portal http://crop-PAL.org. The subcellular localization information housed in cropPAL helps to depict plant cells as compartmentalized protein networks that can be investigated for improving crop yield and quality, and developing new biotechnological solutions to agricultural challenges.

Defence Signalling Pathways Involved in Plant Resistance and Phosphite-Mediated Control of Phytophthora Cinnamomi

Phytophthora cinnamomi is one of the most devastating plant pathogens worldwide. Current control of P. cinnamomi in natural ecosystems primarily relies on chemical phosphite (Phi). To investigate host- and Phi-mediated resistance, A. thaliana ecotypes and mutants defective in salicylic acid (SA), jasmonic acid (JA), ethylene (ET) and abscisic acid (ABA) signalling pathways were screened for susceptibility to P. cinnamomi. In contrast to Col-0, the aba2-4 mutant, deficient in the synthesis of ABA, was susceptible suggesting a role for ABA in resistance to P. cinnamomi. Phi treatment increased resistance in aba2-4, but not to the level of Col-0, suggesting that Phi may act through both ABA-dependent and ABA-independent pathways. Phi treatment or P. cinnamomi inoculation of Col-0 down-regulated AtMYC2, a positive regulator of ABA signalling, which negatively regulates JA/ET-related pathogenesis-related genes, such as PDF1.2, whilst positively regulating JA-mediated herbivore responses such as VSP and PI. Consistent with this, P. cinnamomi or Phi treatment caused up-regulation of PDF1.2 and THI2.1 and down-regulation of VSP2 and the ABA-responsive gene RD22. Despite the up-regulation of JA/ET-dependent defence genes, the JA-defective mutant, jar1-1 and ET-defective mutants, ein2-1 and etr1-3, showed wild-type levels of resistance to P. cinnamomi, suggesting that these JA/ET defences are not required for resistance to P. cinnamomi. These results suggest that the resistance response of Col-0 act, at least in part, through a mechanism dependent on ABA synthesis, which appears independent of the interaction between ABA and elements of the JA/ET pathway, whilst Phi-mediated resistance, although inducing a response resembling the resistance response of Col-0, is independent of ABA signalling.

Large-scale density-based screening for pea weevil resistance in advanced backcross lines derived from cultivated field pea (Pisum sativum) and Pisum fulvum

Abstract. The pea weevil, Bruchus pisorum, is one of the most intractable pest problems of cultivated field pea (Pisum sativum) in the world. Pesticide application, either as a contact insecticide spray to the field pea crop or fumigation of the harvested seed, is the only available method for its control. The aim of the study was to develop a quick and reliable method to screen for pea weevil resistance and increase efficiency in breeding for this important trait. Backcrossed progenies derived from an interspecific cross between cultivated field pea and its wild relative (Pisum fulvum, source of resistance for pea weevil) were subjected to natural infestation in field plots. Mature seeds were hand-harvested, stored to allow development of adult beetles, and then separated into infested and non-infested using a density separation method in 30% caesium chloride (CsCl). Susceptibility and resistance of the progenies were calculated based on this method and further confirmed by a glasshouse bioassay. Resistance in backcross populations improved considerably through selection of resistant lines using the density separation method. We found that the method using CsCl separation is a useful tool in breeding for pea weevil resistance. We were able to introgress pea weevil resistance from P. fulvum into cultivated field pea through backcrossing to produce several advanced pea weevil resistant lines following this procedure.

The pentatricopeptide repeat protein EMB2654 is essential for trans-splicing of a chloroplast small ribosomal subunit transcript

Binding of the pentatricopeptide repeat protein EMB2654 to one-half of the plastid rps12 intron is essential for trans-splicing, production of plastid ribosomes, and embryogenesis in Arabidopsis. We report the partial complementation and subsequent comparative molecular analysis of two nonviable mutants impaired in chloroplast translation, one (emb2394) lacking the RPL6 protein, and the other (emb2654) carrying a mutation in a gene encoding a P-class pentatricopeptide repeat protein. We show that EMB2654 is required for the trans-splicing of the plastid rps12 transcript and that therefore the emb2654 mutant lacks Rps12 protein and fails to assemble the small subunit of the plastid ribosome, explaining the loss of plastid translation and consequent embryo-lethal phenotype. Predictions of the EMB2654 binding site match a small RNA “footprint” located on the 5′ half of the trans-spliced intron that is almost absent in the partially complemented mutant. EMB2654 binds sequence specifically to this target sequence in vitro. Altered patterns in nuclease-protected small RNA fragments in emb2654 show that EMB2654 binding must be an early step in, or prior to, the formation of a large protein-RNA complex covering the free ends of the two rps12 intron halves.

A quantitative PCR assay for accurate in planta quantification of the necrotrophic pathogen Phytophthora cinnamomi

A reliable method for measuring disease progression is important when evaluating susceptibility in host—pathogen interactions. We describe a sensitive quantitative polymerase chain reaction (QPCR) assay that enables quantitative measurement of in planta DNA of the necrotrophic pathogen, Phytophthora cinnamomi, that avoids problems caused by variation in DNA extraction efficiency and degradation of host DNA during host tissue necrosis. Normalization of pathogen DNA to sample fresh weight or host DNA in samples with varying degrees of necrosis led to overestimation of pathogen biomass. Purified plasmid DNA, containing the pScFvB1 mouse gene, was added during DNA extraction and pathogen biomass was normalized based on plasmid DNA rather than host DNA or sample fresh weight. This method is robust and improves the accuracy of pathogen measurement in both resistant (non-host A. thaliana–P. cinnamomi) and susceptible (host Lupinus angustifolius–P. cinnamomi) interactions to allow accurate measurement of pathogen biomass even in the presence of substantial host cell necrosis.

Importance of spatial and spectral data reduction in the detection of internal defects in food products

Despite the importance of data reduction as part of the processing of reflection-based classifications, this study represents one of the first in which the effects of both spatial and spectral data reductions on classification accuracies are quantified. Furthermore, the effects of approaches to data reduction were quantified for two separate classification methods, linear discriminant analysis (LDA) and support vector machine (SVM). As the model dataset, reflection data were acquired using a hyperspectral camera in 230 spectral channels from 401 to 879 nm (spectral resolution of 2.1 nm) from field pea (Pisum sativum) samples with and without internal pea weevil (Bruchus pisorum) infestation. We deployed five levels of spatial data reduction (binning) and eight levels of spectral data reduction (40 datasets). Forward stepwise LDA was used to select and include only spectral channels contributing the most to the separation of pixels from non-infested and infested field peas. Classification accuracies obtained with LDA and SVM were based on the classification of independent validation datasets. Overall, SVMs had significantly higher classification accuracies than LDAs (P < 0.01). There was a negative association between pixel resolution and classification accuracy, while spectral binning equivalent to up to 98% data reduction had negligible effect on classification accuracies. This study supports the potential use of reflection-based technologies in the quality control of food products with internal defects, and it highlights that spatial and spectral data reductions can (1) improve classification accuracies, (2) vastly decrease computer constraints, and (3) reduce analytical concerns associated with classifications of large and high-dimensional datasets.

Interspecific introgression of male sterility from tetraploid oilseed Brassica napus to diploid vegetable B. rapa through hybridisation and backcrossing

Abstract. Interspecific F1 hybrids were obtained from a cross between a male sterile Brassica napus (2n = 4x = 38, AA (20) and CC (18) genomes) and an inbreeding line B. rapa (Purple Cai-Tai inbred line 9418, 2n = 2x = 20, AA (20) genome) to introgress male sterility from a tetraploid into a diploid through backcrossing. The morphological characteristics of F1 plants were more like the female parent B. napus and segregated considerably in BC1 when backcrossed to the recurrent parent Purple Cai-Tai. The progeny became stable and more similar to Purple Cai-Tai by BC4. Most C genome chromosomes were found to be eliminated, based on cytogenetic analysis. The majority of chromosomes were eliminated at very early backcross stages, with only 20–26 chromosomes in BC1 plants, and some chromosomes were eliminated gradually with increased backcross generations. The BC4 plants were generally stable with exactly 20 chromosomes. Analysis by AFLP indicated that 49.5–68.7% of the total bands eliminated from F1 to BC4 were female parent specific, and ∼12% of B. napus bands were retained with increased backcrossing. The genetic materials controlling sterility from the female parent B. napus were introgressed successfully into the BC4 plants even though most B. napus chromosomes/genetic materials were eliminated during the backcross process.