Tracking flight activity of potato leafhopper (Hemiptera: Cicadellidae) with the Midwest Suction Trap Network.

Potato leafhopper (PLH), Empoasca fabae Harris (Hemiptera: Cicadellidae), is an economic pest of a variety of crops that migrates between overwintering sites in the southern United States and northern breeding grounds. Since 2005, the Midwest Suction Trap Network (STN) has monitored the magnitude and timing of aerially dispersing aphids' activity, but the potential of the network to monitor other taxa is only beginning to be explored. Here, we use the Midwest STN to examine how the magnitude and timing of PLH activity vary with weather, cropland cover, and time of year. We found that weekly PLH activity increased early in the season (May-June) with increasing degree day accumulation and decreased mid-season (July-August) with increasing occurrence of rain. The first detections occurred earlier in southern latitudes, while the last detections occurred sooner, when there was more surrounding potato land cover, and later over time between 2018 and 2021 and in southern latitudes. PLH activity was thus longer in duration in southern latitudes and has continued to extend later into the year overall. Resolving uncertainty about how well the Midwest STN captures migratory activity and how closely suction trap detections reflect local population densities in crop fields remain important research priorities before the potential of the Midwest STN for PLH monitoring can be realized. Still, observed patterns suggest that PLH could increase in economic importance as insects disperse over larger portions of the growing season in the warming, agriculturally productive US Midwest and that the STN can become a useful tool to monitor these changes.

Remarkably High Internal Transcribed Spacer Haplotype Diversity of the Fungal Select Agent Coniothyrium glycines Discovered Throughout Its Range in Sub-Saharan Africa.

Red leaf blotch of soybean, caused by the fungus Coniothyrium glycines, is a foliar disease characterized by blotching, necrosis, and defoliation that has only been reported from Africa. The species is listed as a Select Agent by the Federal Select Agent Program due to its potentially devastating impacts to soybean production should it spread to the United States. Despite its potential import, very few isolates are available for study. Herein, we obtained 96 new C. glycines isolates from six soybean-producing countries throughout sub-Saharan Africa. Along with 12 previously collected ones, we sequenced each at the internal transcribed spacer (ITS) region. Between all isolates, we identified a total of 28 single-nucleotide polymorphisms and 23 haplotypes. One hypothesis to explain the tremendous diversity uncovered at the ITS-which is generally conserved within a species-is that our current species concept of C. glycines is too broad and that there may be multiple species that cause red leaf blotch. Zambia contained the highest haplotype diversity, a significant fraction of which remains unsampled. Most haplotypes were specific to a single country, except for two, which were found in Zambia and either neighboring Mozambique or Zimbabwe. This geographic specificity indicates that the ITS region may be useful for identifying source populations or routes of transmission should this pathogen spread beyond Africa. The observed geographic partitioning of this pathogen is likely the result of millions of years of replication on little-studied native hosts, given that soybean has only been cultivated in Africa since the early 1900s.

An NBS-LRR protein in the Rpp1 locus negates the dominance of Rpp1-mediated resistance against Phakopsora pachyrhizi in soybean.

The soybean Rpp1 locus confers resistance to Phakopsora pachyrhizi, causal agent of rust, and resistance is usually dominant over susceptibility. However, dominance of Rpp1-mediated resistance is lost when a resistant genotype (Rpp1 or Rpp1b) is crossed with susceptible line TMG06_0011, and the mechanism of this dominant susceptibility (DS) is unknown. Sequencing the Rpp1 region reveals that the TMG06_0011 Rpp1 locus has a single nucleotide-binding site leucine-rich repeat (NBS-LRR) gene (DS-R), whereas resistant PI 594760B (Rpp1b) is similar to PI 200492 (Rpp1) and has three NBS-LRR resistance gene candidates. Evidence that DS-R is the cause of DS was reflected in virus-induced gene silencing of DS-R in Rpp1b/DS-R or Rpp1/DS-R heterozygous plants with resistance partially restored. In heterozygous Rpp1b/DS-R plants, expression of Rpp1b candidate genes was not significantly altered, indicating no effect of DS-R on transcription. Physical interaction of the DS-R protein with candidate Rpp1b resistance proteins was supported by yeast two-hybrid studies and in silico modeling. Thus, we conclude that suppression of resistance most likely does not occur at the transcript level, but instead probably at the protein level, possibly with Rpp1 function inhibited by binding to the DS-R protein. The DS-R gene was found in other soybean lines, with an estimated allele frequency of 6% in a diverse population, and also found in wild soybean (Glycine soja). The identification of a dominant susceptible NBS-LRR gene provides insight into the behavior of NBS-LRR proteins and serves as a reminder to breeders that the dominance of an R gene can be influenced by a susceptibility allele.

The Role of a Soybean 14-3-3 Gene (Glyma05g29080) on White Mold Resistance and Nodulation Investigations Using CRISPR-Cas9 Editing and RNA Silencing.

The role of a soybean 14-3-3 gene (Glyma05g29080) in defense against white mold and in nodulation was investigated by loss-of-gene-function with CRISPR-Cas9 editing and silencing of RNA interference (RNAi). Particle bombardment was used to introduce the CRISPR expression cassette to target the soybean 14-3-3 gene and an RNAi construct to silence gene transcription. Transmission of the edited 14-3-3 gene and the RNAi construct was confirmed in their respective progeny. The recovered transgenic plants and their progeny were significantly more susceptible to Sclerotinia sclerotiorum infection and showed a significant reduction in nodulation, thus confirming the role of the 14-3-3 gene (Glyma05g29080) in both nodulation and defense.

Characterization and analysis of the promoter region of monodehydroascorbate reductase 4 (CpMDAR4) in papaya.

KEY MESSAGE: Differential spatial and temporal expression patterns due to regulatory cis-elements and two different isoforms are detected among CpMDAR4 alleles in papaya. The aim of this research was to study the effects of cis-element differences between the X, Y and Yh alleles on the expression of CpMDAR4, a potential candidate gene for sex differentiation in papaya, using a transcriptional reporter system in a model species Arabidopsis thaliana. Possible effects of a retrotransposon insertion in the Y and Yh alleles on the transcription and expression of CpMDAR4 alleles in papaya flowers were also examined. When comparing promoters and cis-regulatory elements among genes in the non-recombining region of the sex chromosomes, paired genes exhibited differences. Our results showed that differences in the promoter sequences of the CpMDAR4 alleles drove the expression of a reporter gene to different flower tissues in Arabidopsis. ß-glucuronidase staining analysis of T2 and T3 lines for constructs containing 5' deletions of native Y and Yh allele promoters showed the loss of specific expression of the reporter gene in the anthers, confirming the existence and location of cis-regulatory element POLLEN1LELAT52. The expression analysis of CpMDAR4 alleles in papaya flowers also showed that all alleles are actively expressed in different flower tissues, with the existence of a shorter truncated isoform, with unknown function, for the Y and Yh alleles due to an LTR-RT insertion in the Y and Yh chromosomes. The observed expression patterns in Arabidopsis thaliana flowers and the expression patterns of CpMDAR4 alleles in papaya flowers suggest that MDAR4 might have a role on development of reproductive organs in papaya, and that it constitutes an important candidate for sex differentiation.

Transcriptome Profiling Reveals Molecular Players in Early Soybean-Sclerotinia sclerotiorum Interaction.

Sclerotinia sclerotiorum causes Sclerotinia stem rot on soybean. Using RNA sequencing, the transcriptomes of the soybean host and the S. sclerotiorum pathogen were simultaneously determined at 4 and 8 h postinoculation (hpi). Two soybean genotypes were involved: a resistant oxalate oxidase (OxO)-transgenic line and its susceptible parent, AC Colibri (AC). Of the 594 genes that were significantly induced by S. sclerotiorum, both hosts expressed genes related to jasmonic acid, ethylene, oxidative burst, and phenylpropanoids. In all, 36% of the differentially expressed genes encoded genes associated with transcription factors, ubiquitination, or general signaling transduction such as receptor-like kinases, mitogen-activated protein kinase kinases, and hormones. No significant differentially expressed genes were identified between genotypes, suggesting that oxalic acid (OA) did not play a differential role in early disease development or primary lesion formation under the conditions used. Looking at pathogen behavior through its gene expression during infection, thousands of genes in S. sclerotiorum were induced at 8 hpi, compared with expression in culture. Many plant cell-wall-degrading enzymes (PCWDEs), sugar transport genes, and genes involved in secondary metabolism were upregulated and could contribute to early pathogenesis. When infecting the OxO plants, there was a higher induction of genes encoding OA, botcinic acid, PCWDEs, proteases, and potential effectors, revealing the wealth of virulence factors available to this pathogen as it attempts to colonize a host. Data presented identify hundreds of genes associated with the very early stages of infection for both the host and pathogen.

Characterization and Comparison of Intestinal Bacterial Microbiomes of Euschistus heros and Piezodorus guildinii Collected in Brazil and the United States.

Background: Herbivorous insects are one of the main biological threats to crops. One such group of insects, stink bugs, do not eat large amounts of tissue when feeding on soybean, but are damaging to the quality of the seed yield as they feed on green developing seeds leading to poorly marketable harvests. In addition to causing physical damage during sucking-feeding activities, the insects can also transmit microbial pathogens, leading to even greater yield loss. Conducting surveys of the insect intestinal microbiome can help identify possible pathogens, as well as detail what healthy stink bug digestive systems have in common. Methods: We used the conserved V4 region of the 16S rRNA gene to characterize the bacterial microbiome of the red-banded stink bug Piezodorus guildinii collected in Brazil and the United States, as well as the neotropical brown stink bug Euschistus heros collected in Brazil. Results: After quality filtering of the data, 192 samples were kept for analyses: 117 samples from P. guildinii covering three sites in Brazil and four sites in the United States, and 75 samples for E. heros covering 10 sites in Brazil. The most interesting observations were that the diversity and abundance of some bacterial families were different in the different ecoregions of Brazil and the United States. Conclusion: Some families, such as Acetobacteraceae, Bacillaceae, Moraxellaceae, Enterobacteriaceae, and Rhodocyclaceae, may be related to the better adaptation in some localities in providing nutrients, break down cellulose, detoxify phytochemicals, and degrade organic compounds, which makes it difficult to control these species.

Genome-wide expression of low temperature response genes in Rosa hybrida L.

Plants respond to low temperature stress during cold acclimation, a complex process involving changes in physiological and biochemical modifications. The rose serves as a good model to investigate low temperature responses in perennial ornamentals. In this study, a heterologous apple microarray is used to investigate genome-wide expression profiles in Rosa hybrida subjected to low temperature dark treatment. Transcriptome profiles are determined in floral buds at 0h, 2h, and 12h of low temperature treatment (4 °C). It is observed that a total of 134 transcripts are up-regulated and 169 transcripts are down-regulated in response to low temperature. Interestingly, a total of eight up-regulated genes, including those coding for two cytochrome P450 proteins, two ankyrin repeat family proteins, two metal ion binding proteins, and two zinc finger protein-related transcription factors, along with a single down-regulated gene, coding for a dynamin-like protein, are detected. Transcript profiles of 12 genes known to be involved in cold stress response are also validated using qRT-PCR. Furthermore, expression patterns of the AP2/ERF gene family of transcription factors are investigated in both floral buds and leaves. Overall, AP2/ERFs genes are more rapidly induced in leaves than in floral buds. Moreover, differential expression of several AP2/ERF genes are detected earlier in vegetative rather than in reproductive tissues. These findings highlight important roles of various low temperature response genes in mediating cold acclimation, thereby allowing roses to adapt to low temperatures, but without adversely affecting flower bud development and subsequent flowering, while vegetative tissues undergo early adaptation to low temperatures.

Population Genomics of the Neotropical Brown Stink Bug, Euschistus heros: The Most Important Emerging Insect Pest to Soybean in Brazil.

Recent changes in soybean management like the adoption of transgenic crops and no-till farming, in addition to the expansion of cultivated areas into new virgin frontiers, are some of the hypotheses that can explain the rise of secondary pests, such as the Neotropical brown stink bug, Euschistus heros, in Brazil. To better access the risk of increased pests like E. heros and to determine probabilities for insecticide resistance spreading, it is necessary first to access the levels of the genetic diversity, how the genetic diversity is distributed, and how natural selection is acting upon the natural variation. Using the genotyping by sequencing (GBS) technique, we generated ~60,000 single-nucleotide polymorphisms (SNPs) distributed across the E. heros genome to answer some of those questions. The SNP data was used to investigate the pattern of genetic structure, hybridization and natural selection of this emerging pest. We found that E. heros populations presented similar levels of genetic diversity with slightly higher values at several central locations in Brazil. Our results also showed strong genetic structure separating northern and southern Brazilian regions (FST = 0.22; p-value = 0.000) with a very distinct hybrid zone at the central region. The analyses also suggest the possibility that GABA channels and odorant receptors might play a role in the process of natural selection. At least one marker was associated with soybean and beans crops, but no association between allele frequency and cotton was found. We discuss the implications of these findings in the management of emerging pests in agriculture, particularly in the context of large areas of monoculture such as soybean and cotton.

Patterns of Genome-Wide Variation, Population Differentiation and SNP Discovery of the Red Banded Stink Bug (Piezodorus guildinii).

Unravelling the details of range expansion and ecological dominance shifts of insect pests has been challenging due to the lack of basic knowledge about population structure, gene flow, and most importantly, how natural selection is affecting the adaptive process. Piezodous guildinii is an emerging pest of soybean in the southern region of the United States, and increasingly important in Brazil in recent years. However, the reasons P. guildinii is gradually becoming more of a problem are questions still mostly unanswered. Here, we have genotyped P. guildinii samples and discovered 1,337 loci containing 4,083 variant sites SNPs that were used to estimate genetic structure and to identify gene candidates under natural selection. Our results revealed the existence of a significant genetic structure separating populations according to their broad geographic origin, i.e., U.S. and Brazil, supported by AMOVA (FGT = 0.26), STRUCTURE, PCA, and FST analyses. High levels of gene flow or coancestry within groups (i.e., within countries) can be inferred from the data, and no spatial pattern was apparent at the finer scale in Brazil. Samples from different seasons show more heterogeneous compositions suggesting mixed ancestry and a more complex dynamic. Lastly, we were able to detect and successfully annotated 123 GBS loci (10.5%) under positive selection. The gene ontology (GO) analysis implicated candidate genes under selection with genome reorganization, neuropeptides, and energy mobilization. We discuss how these findings could be related to recent outbreaks and suggest how new efforts directed to better understand P. guildinii population dynamics.

Diatraea saccharalis history of colonization in the Americas. The case for human-mediated dispersal.

The sugarcane borer moth, Diatraea saccharalis, is one of the most important pests of sugarcane and maize crops in the Western Hemisphere. The pest is widespread throughout South and Central America, the Caribbean region and the southern United States. One of the most intriguing features of D. saccharalis population dynamics is the high rate of range expansion reported in recent years. To shed light on the history of colonization of D. saccharalis, we investigated the genetic structure and diversity in American populations using single nucleotide polymorphism (SNPs) markers throughout the genome and sequences of the mitochondrial gene cytochrome oxidase (COI). Our primary goal was to propose possible dispersal routes from the putative center of origin that can explain the spatial pattern of genetic diversity. Our findings showed a clear correspondence between genetic structure and the geographical distributions of this pest insect on the American continents. The clustering analyses indicated three distinct groups: one composed of Brazilian populations, a second group composed of populations from El Salvador, Mexico, Texas and Louisiana and a third group composed of the Florida population. The predicted time of divergence predates the agriculture expansion period, but the pattern of distribution of haplotype diversity suggests that human-mediated movement was most likely the factor responsible for the widespread distribution in the Americas. The study of the early history of D. saccharalis promotes a better understanding of range expansion, the history of invasion, and demographic patterns of pest populations in the Americas.

Morphological and molecular characterization of Brazilian populations of Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) and the evolutionary relationship among species of Diatraea Guilding.

The sugarcane borer or corn stalk borer, Diatraea Guilding is polyphagous insect pest of many important crops such as corn, sorghum and sugarcane. Losses arising from the attack of Diatraea species have been a serious problem, which may cause loss in sugarcane production around 0.25% in sugar, 0.20% in alcohol and 0.77% of body weight for every 1% infestation and up to 21% in corn production fields. In Brazil, the most commonly reported species are Diatraea saccharalis (Fabricius, 1794) and Diatraea impersonatella (Walker, 1863) (= D. flavipennella). However, multiple other species of Diatraea have been identified in Brazil according to the literature. Currently, little information exists on the presence of the other species causing injury to sugarcane and corn. The objectives of this study were to improve the accuracy of species assignment, evaluate the population genetic structure, and address many of the outstanding questions of systematics and evolution of Brazilian populations of D. saccharalis. To address these main questions, classical taxonomic methods were used, focused on morphological characterization of the reproductive organs, especially the male genitalia. In addition, genetic studies were performed using simple sequence repeats (SSR) and a fragment of cytochrome C oxidase subunit I (COI) gene. The data and findings from this research will contribute to the understanding of evolutionary aspects of insect pests in order to develop more effective and sustainable population management practices.

Genome-wide association mapping of resistance to a Brazilian isolate of Sclerotinia sclerotiorum in soybean genotypes mostly from Brazil.

BACKGROUND: Sclerotinia Stem Rot (SSR), caused by the fungal pathogen Sclerotinia sclerotiorum, is ubiquitous in cooler climates where soybean crops are grown. Breeding for resistance to SSR remains challenging in crops like soybean, where no single gene provides strong resistance, but instead, multiple genes work together to provide partial resistance. In this study, a genome-wide association study (GWAS) was performed to dissect the complex genetic architecture of soybean quantitative resistance to SSR and to provide effective molecular markers that could be used in breeding programs. A collection of 420 soybean genotypes were selected based on either reports of resistance, or from one of three different breeding programs in Brazil, two commercial, one public. Plant genotype sensitivity to SSR was evaluated by the cut stem inoculation method, and lesion lengths were measured at 4 days post inoculation. RESULTS: Genotyping-by-sequencing was conducted to genotype the 420 soybean lines. The TASSEL 5 GBSv2 pipeline was used to call SNPs under optimized parameters, and with the extra step of trimming adapter sequences. After filtering missing data, heterozygosity, and minor allele frequency, a total of 11,811 SNPs and 275 soybean genotypes were obtained for association analyses. Using a threshold of FDR-adjusted p-values <0.1, the Compressed Mixed Linear Model (CMLM) with Genome Association and Prediction Integrated Tool (GAPIT), and the Fixed and Random Model Circulating Probability Unification (FarmCPU) methods, both approaches identified SNPs with significant association to disease response on chromosomes 1, 11, and 18. The CMLM also found significance on chromosome 19, whereas FarmCPU also identified significance on chromosomes 4, 9, and 16. CONCLUSIONS: These similar and yet different results show that the computational methods used can impact SNP associations in soybean, a plant with a high degree of linkage disequilibrium, and in SSR resistance, a trait that has a complex genetic basis. A total of 125 genes were located within linkage disequilibrium of the three loci shared between the two models. Their annotations and gene expressions in previous studies of soybean infected with S. sclerotiorum were examined to narrow down the candidates.

Investigating the Control of Chlorophyll Degradation by Genomic Correlation Mining.

Chlorophyll degradation is an intricate process that is critical in a variety of plant tissues at different times during the plant life cycle. Many of the photoactive chlorophyll degradation intermediates are exceptionally cytotoxic necessitating that the pathway be carefully coordinated and regulated. The primary regulatory step in the chlorophyll degradation pathway involves the enzyme pheophorbide a oxygenase (PAO), which oxidizes the chlorophyll intermediate pheophorbide a, that is eventually converted to non-fluorescent chlorophyll catabolites. There is evidence that PAO is differentially regulated across different environmental and developmental conditions with both transcriptional and post-transcriptional components, but the involved regulatory elements are uncertain or unknown. We hypothesized that transcription factors modulate PAO expression across different environmental conditions, such as cold and drought, as well as during developmental transitions to leaf senescence and maturation of green seeds. To test these hypotheses, several sets of Arabidopsis genomic and bioinformatic experiments were investigated and re-analyzed using computational approaches. PAO expression was compared across varied environmental conditions in the three separate datasets using regression modeling and correlation mining to identify gene elements co-expressed with PAO. Their functions were investigated as candidate upstream transcription factors or other regulatory elements that may regulate PAO expression. PAO transcript expression was found to be significantly up-regulated in warm conditions, during leaf senescence, and in drought conditions, and in all three conditions significantly positively correlated with expression of transcription factor Arabidopsis thaliana activating factor 1 (ATAF1), suggesting that ATAF1 is triggered in the plant response to these processes or abiotic stresses and in result up-regulates PAO expression. The proposed regulatory network includes the freezing, senescence, and drought stresses modulating factor ATAF1 and various other transcription factors and pathways, which in turn act to regulate chlorophyll degradation by up-regulating PAO expression.

The role of invertases in plant compensatory responses to simulated herbivory.

BACKGROUND: The ability of a plant to overcome animal-induced damage is referred to as compensation or tolerance and ranges from undercompensation (decreased fitness when damaged) to overcompensation (increased fitness when damaged). Although it is clear that genetic variation for compensation exists among plants, little is known about the specific genetic underpinnings leading to enhanced fitness. Our previous study identified the enzyme GLUCOSE-6-PHOSPHATE DEHYDROGENASE 1 (G6PD1) as a key regulator contributing to the phenomenon of overcompensation via its role in the oxidative pentose phosphate pathway (OPPP). Apart from G6PD1 we also identified an invertase gene which was up-regulated following damage and that potentially integrates with the OPPP. The invertase family of enzymes hydrolyze sucrose to glucose and fructose, whereby the glucose produced is shunted into the OPPP and presumably supports plant regrowth, development, and ultimately compensation. In the current study, we measured the relative expression of 12 invertase genes over the course of plant development in the Arabidopsis thaliana genotypes Columbia-4 and Landsberg erecta, which typically overcompensate and undercompensate, respectively, when damaged. We also compared the compensatory performances of a set of invertase knockout mutants to the Columbia-4 wild type. RESULTS: We report that Columbia-4 significantly up-regulated 9 of 12 invertase genes when damaged relative to when undamaged, and ultimately overcompensated for fruit production. Landsberg erecta, in contrast, down-regulated two invertase genes following damage and suffered reduced fitness. Knockout mutants of two invertase genes both exhibited significant undercompensation for fruit production, exhibiting a complete reversal of the wild type Col-4's overcompensation. CONCLUSION: Collectively, these results confirm that invertases are essential for not only normal plant growth and development, but also plants' abilities to regrow and ultimately compensate for fitness following apical damage.

Genomic evaluation of oxalate-degrading transgenic soybean in response to Sclerotinia sclerotiorum infection.

Oxalate oxidases (OxO) catalyse the degradation of oxalic acid (OA). Highly resistant transgenic soybean carrying an OxO gene and its susceptible parent soybean line, AC Colibri, were tested for genome-wide gene expression in response to the necrotrophic, OA-producing pathogen Sclerotinia sclerotiorum using soybean cDNA microarrays. The genes with changed expression at statistically significant levels (overall F-test P-value cut-off of 0.0001) were classified into functional categories and pathways, and were analysed to evaluate the differences in transcriptome profiles. Although many genes and pathways were found to be similarly activated or repressed in both genotypes after inoculation with S. sclerotiorum, the OxO genotype displayed a measurably faster induction of basal defence responses, as observed by the differential changes in defence-related and secondary metabolite genes compared with its susceptible parent AC Colibri. In addition, the experiment presented provides data on several other transcripts that support the hypothesis that S. sclerotiorum at least partially elicits the hypersensitive response, induces lignin synthesis (cinnamoyl CoA reductase) and elicits as yet unstudied signalling pathways (G-protein-coupled receptor and related). Of the nine genes showing the most extreme opposite directions of expression between genotypes, eight were related to photosynthesis and/or oxidation, highlighting the importance of redox in the control of this pathogen.

Transcriptome analyses suggest a disturbance of iron homeostasis in soybean leaves during white mould disease establishment.

Sclerotinia sclerotiorum is a serious pathogen of numerous crops around the world. The major virulence factor of this pathogen is oxalic acid (OA). Mutants that cannot produce OA do not cause disease, and plants that express enzymes that degrade OA, such as oxalate oxidase (OxO), are very resistant to S. sclerotiorum. To examine the effect of OA on plants, we infiltrated soybean leaves with 5 mm OA and examined the gene expression changes at 2 h post-infiltration. By comparing the gene expression levels between leaves of a transgenic soybean carrying an OxO gene (OxO) and its parent AC Colibri (AC) infiltrated with OA (pH 2.4) or water (pH 2.4 or 5.5), we were able to compare the effects of OA dependent or independent of its pH. Gene expression by microarray analysis identified 2390 genes that showed changes in expression, as determined using an overall F-test P-value cut-off of 0.001. The additional requirement that at least one pairwise t-test false discovery rate (FDR)-corrected P value should be less than 0.001 reduced the list of the most highly significant differentially expressed genes to 1054. Independent of pH, OA altered the expression levels of 78 genes, with ferritin showing the strongest induction by OA. The combination of OA plus its low pH caused 1045 genes (99% of all significant genes) to be differentially expressed, with many of the up-regulated genes being related to basal defence, such as genes of the phenylpropanoid pathway and various cytochrome P450s. RNA-seq was also conducted on four samples: OxO and AC genotypes infiltrated with either OA pH 2.4 or water pH 2.4. The RNA-seq analysis also identified ferritin paralogues as being strongly induced by OA. As the expression of ferritin, a gene that encodes for an iron storage protein, is induced by free iron, these results suggest that S. sclerotiorum benefits from the ability of OA to free iron from plant proteins, as this induces host cell death, and also allows the uptake and assimilation of the iron for its own metabolic needs.

Effect of Fusarium virguliforme phytotoxin on soybean gene expression suggests a role in multidimensional defence.

Sudden death syndrome (SDS), caused by Fusarium virguliforme, is an important yield-limiting disease of soybean. This soil-borne fungus colonizes soybean roots causing root rot, and also releases a phytotoxin that is translocated to leaves causing interveinal chlorosis and necrosis leading to defoliation and early maturation. The objective of our study was to compare gene expression profiles during the early response of soybean leaves exposed to sterile culture filtrates of F. virguliforme in soybean genotypes with different levels of resistance to SDS. The analysis identified SDS-related defence genes that were induced in the most resistant genotype, but not in the other genotypes. Further functional annotations based on sequence homology suggested that some of the induced genes probably encode proteins involved in cell wall modification, detoxification, defence responses, primary metabolism and membrane transport. Quantitative real-time reverse-transcribed polymerase chain reaction confirmed the differential transcript accumulation of a subset of these genes. In addition, in silico mapping of differentially expressed genes to SDS-resistant quantitative trait loci allowed for the identification of new potential defence genes that could be genetically mapped to the soybean genome, and could be used further in a marker-assisted selection programme. A comparison of the response of soybean to F. virguliforme phytotoxin (Fv toxin) relative to other biotic and abiotic stresses revealed that the resistance response to Fv toxin is quite similar to the response to inoculation with an incompatible Pseudomonas syringae pv. glycinea strain, suggesting that Fv toxin might induce hypersensitive response pathways in soybean leaf tissues in the absence of pathogen in these tissues.

Divergent patterns of endogenous small RNA populations from seed and vegetative tissues of Glycine max.

BACKGROUND: Small non-coding RNAs (smRNAs) are known to have major roles in gene regulation in eukaryotes. In plants, knowledge of the biogenesis and mechanisms of action of smRNA classes including microRNAs (miRNAs), short interfering RNAs (siRNAs), and trans-acting siRNAs (tasiRNAs) has been gained mostly through studies with Arabidopsis. In recent years, high throughput sequencing of smRNA populations has enabled extension of knowledge from model systems to plants with larger, more complex genomes. Soybean (Glycine max) now has many genomics resources available including a complete genome sequence and predicted gene models. Relatively little is known, however, about the full complement of its endogenous smRNAs populations and the silenced genes. RESULTS: Using Illumina sequencing and computational analysis, we characterized eight smRNA populations from multiple tissues and organs of soybean including developing seed and vegetative tissues. A total of 41 million raw sequence reads collapsed into 135,055 unique reads were mapped to the soybean genome and its predicted cDNA gene models. Bioinformatic analyses were used to distinguish miRNAs and siRNAs and to determine their genomic origins and potential target genes. In addition, we identified two soybean TAS3 gene homologs, the miRNAs that putatively guide cleavage of their transcripts, and the derived tasiRNAs that could target soybean genes annotated as auxin response factors. Tissue-differential expression based on the flux of normalized miRNA and siRNA abundances in the eight smRNA libraries was evident, some of which was confirmed by smRNA blotting. Our global view of these smRNA populations also revealed that the size classes of smRNAs varied amongst different tissues, with the developing seed and seed coat having greater numbers of unique smRNAs of the 24-nt class compared to the vegetative tissues of germinating seedlings. The 24-nt class is known to be derived from repetitive elements including transposons. Detailed analysis of the size classes associated with ribosomal RNAs and transposable element families showed greater diversity of smRNAs in the 22- and 24-nt size classes. CONCLUSIONS: The flux of endogenous smRNAs within multiple stages and tissues of seed development was contrasted with vegetative tissues of soybean, one of the dominant sources of protein and oil in world markets. The smRNAs varied in size class, complexity of origins, and possible targets. Sequencing revealed tissue-preferential expression for certain smRNAs and expression differences among closely related miRNA family members.

14-3-3 proteins SGF14c and SGF14l play critical roles during soybean nodulation.

The soybean (Glycine max) genome contains 18 members of the 14-3-3 protein family, but little is known about their association with specific phenotypes. Here, we report that the Glyma0529080 Soybean G-box Factor 14-3-3c (SGF14c) and Glyma08g12220 (SGF14l) genes, encoding 14-3-3 proteins, appear to play essential roles in soybean nodulation. Quantitative reverse transcription-polymerase chain reaction and western-immunoblot analyses showed that SGF14c mRNA and protein levels were specifically increased in abundance in nodulated soybean roots 10, 12, 16, and 20 d after inoculation with Bradyrhizobium japonicum. To investigate the role of SGF14c during soybean nodulation, RNA interference was employed to silence SGF14c expression in soybean roots using Agrobacterium rhizogenes-mediated root transformation. Due to the paleopolyploid nature of soybean, designing a specific RNA interference sequence that exclusively targeted SGF14c was not possible. Therefore, two highly similar paralogs (SGF14c and SGF14l) that have been shown to function as dimers were silenced. Transcriptomic and proteomic analyses showed that mRNA and protein levels were significantly reduced in the SGF14c/SGF14l-silenced roots, and these roots exhibited reduced numbers of mature nodules. In addition, SGF14c/SGF14l-silenced roots contained large numbers of arrested nodule primordia following B. japonicum inoculation. Transmission electron microscopy further revealed that the host cytoplasm and membranes, except the symbiosome membrane, were severely degraded in the failed nodules. Altogether, transcriptomic, proteomic, and cytological data suggest a critical role of one or both of these 14-3-3 proteins in early development stages of soybean nodules.