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

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

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Not AvailableFusarium 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. virguliforme infection. 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.USDA NIFAUnited States Department of Agriculture (USDA) [2013 - 68004 - 20374]Iowa Soybean Association; National Institute of Food and Agriculture (NIFA), United States Department of Agriculture [2013 - 68004 - 20374

Reduced expression levels of soybean genes following <i>F</i>. <i>virguliforme</i> infection as compared to the water control.

<p>A. Expression levels of four selected soybean genes following water treatment at early (S1: 3 and 5 d) and late time (S2: 10 and 24 d) periods and <i>F</i>. <i>virguliforme</i> infection at early (S3: 3 and 5 d) and late time (S4: 10 and 24 d) periods. B. RT-PCR analyses of the selected soybean genes. RT-PCR products of each of the four selected soybean amplified from RNAs of root tissues harvested 8 and 12 h, and 1, 2, 3, 5 days following (i) water treatment or (ii) infection with the <i>F</i>. <i>virguliforme</i> Mont-1. The results presented here are from one of three independent experiments showing similar results. <i>Glyma12g12470</i> is from the Glyma.Wm82.a1.v1.1 version of the soybean genome sequence. Other three genes are from the recent version of the soybean genome sequence (Glyma.Wm82.a2v1). <i>Elf1b</i>, elongation factor 1-β encoded by <i>Glyma02g44460</i>. C–F. Quantified expression levels of four selected genes. Gel pictures of the three biological replications are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163106#pone.0163106.s008" target="_blank">S8 Fig</a>.</p

Distribution of differentially expressed genes in soybean roots in response to <i>F</i>. <i>virguliforme</i> infection.

<p>A. Total number of genes differentially (with FC ≥ 10) regulated by <i>F</i>. <i>virguliforme</i> infection. B. Number of genes up-regulated in the infected roots at early and late time-periods. C. Number of genes repressed in the infected roots at early and late time-periods. S1, pooled RNA samples prepared from roots harvested 3 and 5 days following water treatment; S2, pooled RNA samples prepared from roots harvested 10 and 24 days following water treatment; S3, pooled RNA samples prepared from roots harvested 3 and 5 days following <i>F</i>. <i>virguliforme</i> infection; S4, pooled RNA samples prepared from roots harvested 10 and 24 days following <i>F</i>. <i>virguliforme</i> infection.</p

Average <i>GmARP1</i> transgene copy numbers among the transgenic lines.

<p>Average <i>GmARP1</i> transgene copy numbers among the transgenic lines.</p

Expression of <i>GmARP1</i> enhances SDS resistance in transgenic soybean plants.

<p>R₁ plants were tested for resistance to <i>F</i>. <i>virguliforme</i> under growth chamber conditions. A. Root phenotype of a resistant (R) and a susceptible (S) R₁ progeny of a transformant, Prom2-ARP1-7, carrying the <i>Prom2-GmARP1</i> fusion gene. B. Enhanced foliar SDS resistance among R₁ progenies. W82, the SDS susceptible line Williams 82; MN1606, the SDS resistant line. C. Chlorophyll content per individual R₁ progeny carrying <i>GmARP1</i> of three independent transformants. ‘Resistant’ and ‘Susceptible’ classes are defined as in (A). D. Average root weight of R₁ progeny of three independent transformants. ‘Resistant’ and ‘Susceptible’ classes are defined as in A. E. Enhanced root resistance among R₁ progenies. Extent of root resistance to the pathogen was expressed in percent; e.g., 100%, healthy roots with no obvious blackening caused by necrosis and rotting due to infection of <i>F</i>. <i>virguliforme</i>. F. Expression of <i>GmARP1</i> transgenes. Two random SDS resistant and susceptible R₁ progenies from each R₀ line were analyzed. Top panel, resistant plants (two representatives from each line). Bottom panel, susceptible plants (two representatives from each line). Red arrow, <i>GmARP1</i>; black arrow, <i>ELF1b</i> internal control. *, significantly different at <i>p<0</i>.<i>01</i>. Results are means ±SE of three independent experiments.</p

Genes down-regulated at late time period in soybean roots infected with <i>F</i>. <i>virguliforme</i>.

<p>Genes down-regulated at late time period in soybean roots infected with <i>F</i>. <i>virguliforme</i>.</p

Genes down-regulated (with FC >10) in soybean roots during early time period following infection with <i>F</i>. <i>virguliforme</i>.

<p>The full list is reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163106#pone.0163106.s014" target="_blank">S2 Dataset</a>.</p