Sequence based polymorphic (SBP) marker technology for targeted genomic regions: its application in generating a molecular map of the Arabidopsis thaliana genome

<p>Abstract</p> <p>Background</p> <p>Molecular markers facilitate both genotype identification, essential for modern animal and plant breeding, and the isolation of genes based on their map positions. Advancements in sequencing technology have made possible the identification of single nucleotide polymorphisms (SNPs) for any genomic regions. Here a sequence based polymorphic (SBP) marker technology for generating molecular markers for targeted genomic regions in Arabidopsis is described.</p> <p>Results</p> <p>A ~3X genome coverage sequence of the <it>Arabidopsis thaliana </it>ecotype, Niederzenz (Nd-0) was obtained by applying Illumina's sequencing by synthesis (Solexa) technology. Comparison of the Nd-0 genome sequence with the assembled Columbia-0 (Col-0) genome sequence identified putative single nucleotide polymorphisms (SNPs) throughout the entire genome. Multiple 75 base pair Nd-0 sequence reads containing SNPs and originating from individual genomic DNA molecules were the basis for developing co-dominant SBP markers. SNPs containing Col-0 sequences, supported by transcript sequences or sequences from multiple BAC clones, were compared to the respective Nd-0 sequences to identify possible restriction endonuclease enzyme site variations. Small amplicons, PCR amplified from both ecotypes, were digested with suitable restriction enzymes and resolved on a gel to reveal the sequence based polymorphisms. By applying this technology, 21 SBP markers for the marker poor regions of the Arabidopsis map representing polymorphisms between Col-0 and Nd-0 ecotypes were generated.</p> <p>Conclusions</p> <p>The SBP marker technology described here allowed the development of molecular markers for targeted genomic regions of Arabidopsis. It should facilitate isolation of co-dominant molecular markers for targeted genomic regions of any animal or plant species, whose genomic sequences have been assembled. This technology will particularly facilitate the development of high density molecular marker maps, essential for cloning genes based on their genetic map positions and identifying tightly linked molecular markers for selecting desirable genotypes in animal and plant breeding experiments.</p

Systemic acquired resistance in soybean is regulated by two proteins, Orthologous to Arabidopsis NPR1

<p>Abstract</p> <p>Background</p> <p>Systemic acquired resistance (SAR) is induced in non-inoculated leaves following infection with certain pathogenic strains. SAR is effective against many pathogens. Salicylic acid (SA) is a signaling molecule of the SAR pathway. The development of SAR is associated with the induction of pathogenesis related (<it>PR</it>) genes. Arabidopsis <it>non-expressor </it>of <it>PR1 </it>(<it>NPR1</it>) is a regulatory gene of the SA signal pathway <abbrgrp><abbr bid="B1">1</abbr><abbr bid="B2">2</abbr><abbr bid="B3">3</abbr></abbrgrp>. SAR in soybean was first reported following infection with <it>Colletotrichum trancatum </it>that causes anthracnose disease. We investigated if SAR in soybean is regulated by a pathway, similar to the one characterized in Arabidopsis.</p> <p>Results</p> <p>Pathogenesis-related gene <it>GmPR1 </it>is induced following treatment of soybean plants with the SAR inducer, 2,6-dichloroisonicotinic acid (INA) or infection with the oomycete pathogen, <it>Phytophthora sojae</it>. In <it>P. sojae</it>-infected plants, SAR was induced against the bacterial pathogen, <it>Pseudomonas syringae </it>pv. glycinea. Soybean <it>GmNPR1-1 </it>and <it>GmNPR1-2 </it>genes showed high identities to Arabidopsis <it>NPR1</it>. They showed similar expression patterns among the organs, studied in this investigation. <it>GmNPR1-1 </it>and <it>GmNPR1-2 </it>are the only soybean homologues of <it>NPR1</it>and are located in homoeologous regions. In <it>GmNPR1-1 </it>and <it>GmNPR1-2 </it>transformed Arabidopsis <it>npr1-1 </it>mutant plants, SAR markers: (i) <it>PR-1 </it>was induced following INA treatment and (ii) <it>BGL2 </it>following infection with <it>Pseudomonas syringae </it>pv. tomato (<it>Pst</it>), and SAR was induced following <it>Pst </it>infection. Of the five cysteine residues, Cys⁸², Cys¹⁵⁰, Cys¹⁵⁵, Cys¹⁶⁰, and Cys²¹⁶involved in oligomer-monomer transition in NPR1, Cys²¹⁶ in GmNPR1-1 and GmNPR1-2 proteins was substituted to Ser and Leu, respectively.</p> <p>Conclusion</p> <p>Complementation analyses in Arabidopsis <it>npr1-1 </it>mutants revealed that homoeologous <it>GmNPR1-1 </it>and <it>GmNPR1-2 </it>genes are orthologous to Arabidopsis <it>NPR1</it>. Therefore, SAR pathway in soybean is most likely regulated by <it>GmNPR1 </it>genes. Substitution of Cys²¹⁶residue, essential for oligomer-monomer transition of Arabidopsis NPR1, with Ser and Leu residues in GmNPR1-1 and GmNPR1-2, respectively, suggested that there may be differences between the regulatory mechanisms of GmNPR1 and Arabidopsis NPR proteins.</p

The Genome Sequence of the Fungal Pathogen Fusarium virguliforme That Causes Sudden Death Syndrome in Soybean

Fusarium virguliforme causes sudden death syndrome (SDS) of soybean, a disease of serious concern throughout most of the soybean producing regions of the world. Despite the global importance, little is known about the pathogenesis mechanisms of F. virguliforme. Thus, we applied Next-Generation DNA Sequencing to reveal the draft F. virguliforme genome sequence and identified putative pathogenicity genes to facilitate discovering the mechanisms used by the pathogen to cause this disease

Tanscriptomic Study of the Soybean-Fusarium virguliforme Interaction Revealed a Novel Ankyrin-Repeat Containing Defense Gene, Expression of Whose during Infection Led to Enhanced Resistance to the Fungal Pathogen in Transgenic Soybean Plants

Fusarium virguliforme causes the serious disease sudden death syndrome (SDS) in soybean. Host resistance to this pathogen is partial and is encoded by a large number of quantitative trait loci, each conditioning small effects. Breeding SDS resistance is therefore challenging and identification of single-gene encoded novel resistance mechanisms is becoming a priority to fight this devastating this fungal pathogen. In this transcriptomic study we identified a few putative soybean defense genes, expression of which is suppressed during F. virguliformeinfection. The F. virguliforme infection-suppressed genes were broadly classified into four major classes. The steady state transcript levels of many of these genes were suppressed to undetectable levels immediately following F. virguliforme infection. One of these classes contains two novel genes encoding ankyrin repeat-containing proteins. Expression of one of these genes, GmARP1, during F. virguliforme infection enhances SDS resistance among the transgenic soybean plants. Our data suggest that GmARP1 is a novel defense gene and the pathogen presumably suppress its expression to establish compatible interaction

A Phosphoproteomics Study of the Soybean root necrosis 1 Mutant Revealed Type II Metacaspases Involved in Cell Death Pathway

The soybean root necrosis 1 (rn1) mutation causes progressive browning of the roots soon after germination and provides increased tolerance to the soil-borne oomycete pathogen Phytophthora sojae in soybean. Toward understanding the molecular basis of the rn1 mutant phenotypes, we conducted tandem mass tag (TMT)-labeling proteomics and phosphoproteomics analyses of the root tissues of the rn1 mutant and progenitor T322 line to identify potential proteins involved in manifestation of the mutant phenotype. We identified 3,160 proteins. When the p-value was set at ≤0.05 and the fold change of protein accumulation between rn1 and T322 at ≥1.5 or ≤0.67, we detected 118 proteins that showed increased levels and 32 proteins decreased levels in rn1 as compared to that in T322. The differentially accumulated proteins (DAPs) are involved in several pathways including cellular processes for processing environmental and genetic information, metabolism and organismal systems. Five pathogenesis-related proteins were accumulated to higher levels in the mutant as compared to that in T322. Several of the DAPs are involved in hormone signaling, redox reaction, signal transduction, and cell wall modification processes activated in plant–pathogen interactions. The phosphoproteomics analysis identified 22 phosphopeptides, the levels of phosphorylation of which were significantly different between rn1 and T322 lines. The phosphorylation levels of two type II metacaspases were reduced in rn1 as compared to T322. Type II metacaspase has been shown to be a negative regulator of hypersensitive cell death. In absence of the functional Rn1 protein, two type II metacaspases exhibited reduced phosphorylation levels and failed to show negative regulatory cell death function in the soybean rn1 mutant. We hypothesize that Rn1 directly or indirectly phosphorylates type II metacaspases to negatively regulate the cell death process in soybean roots

Humidity assay for studying plant-pathogen interactions in miniature controlled discrete humidity environments with good throughput

This paper reports a highly economical and accessible approach to generate different discrete relative humidity conditions in spatially separated wells of a modified multi-well plate for humidity assay of plant-pathogen interactions with good throughput. We demonstrated that a discrete humidity gradient could be formed within a few minutes and maintained over a period of a few days inside the device. The device consisted of a freeway channel in the top layer, multiple compartmented wells in the bottom layer, a water source, and a drying agent source. The combinational effects of evaporation, diffusion, and convection were synergized to establish the stable discrete humidity gradient. The device was employed to study visible and molecular disease phenotypes of soybean in responses to infection by Phytophthora sojae, an oomycete pathogen, under a set of humidity conditions, with two near-isogenic soybean lines, Williams and Williams 82, that differ for a Phytophthora resistance gene (Rps1-k). Our result showed that at 63% relative humidity, the transcript level of the defense gene GmPR1 was at minimum in the susceptible soybean line Williams and at maximal level in the resistant line Williams 82 following P. sojae CC5C infection. In addition, we investigated the effects of environmental temperature, dimensional and geometrical parameters, and other configurational factors on the ability of the device to generate miniature humidity environments. This work represents an exploratory effort to economically and efficiently manipulate humidity environments in a space-limited device and shows a great potential to facilitate humidity assay of plant seed germination and development, pathogen growth, and plant-pathogen interactions. Since the proposed device can be easily made, modified, and operated, it is believed that this present humidity manipulation technology will benefit many laboratories in the area of seed science, plant pathology, and plant-microbe biology, where humidity is an important factor that influences plant disease infection, establishment, and development

On the Significance of Process Comprehension for Conducting Targeted ICS Attacks

The exploitation of Industrial Control Systems (ICSs) has been described as both easy and impossible, where is the truth? Post-Stuxnet works have included a plethora of ICS focused cyber secu- rity research activities, with topics covering device maturity, network protocols, and overall cyber security culture. We often hear the notion of ICSs being highly vulnerable due to a lack of inbuilt security mechanisms, considered a low hanging fruit to a variety of low skilled threat actors. While there is substantial evidence to support such a notion, when considering targeted attacks on ICS, it is hard to believe an attacker with limited resources, such as a script kiddie or hacktivist, using publicly accessible tools and exploits alone, would have adequate knowledge and resources to achieve targeted operational process manipulation, while simultaneously evade detection. Through use of a testbed environment, this paper provides two practical examples based on a Man-In-The-Middle scenario, demonstrating the types of information an attacker would need obtain, collate, and comprehend, in order to begin targeted process manipulation and detection avoidance. This allows for a clearer view of associated challenges, and illustrate why targeted ICS exploitation might not be possible for every malicious actor