Host-derived gene silencing of parasite fitness genes improves resistance to soybean cyst nematodes in stable transgenic soybean.

KEY MESSAGE: Soybean expressing small interfering RNA of SCN improved plant resistance to SCN consistently, and small RNA-seq analysis revealed a threshold of siRNA expression required for resistance ability. Soybean cyst nematode (SCN), Heterodera glycines, is one of the most destructive pests limiting soybean production worldwide, with estimated losses of $1 billion dollars annually in the USA alone. RNA interference (RNAi) has become a powerful tool for silencing gene expression. We report here that the expression of hairpin RNAi constructs, derived from two SCN genes related to reproduction and fitness, HgY25 and HgPrp17, enhances resistance to SCN in stably transformed soybean plants. The analyses of T3 to T5 generations of stable transgenic soybeans by molecular strategies and next-generation sequencing confirmed the presence of specific short interfering RNAs complementary to the target SCN genes. Bioassays performed on transgenic soybean lines targeting SCN HgY25 and HgPrp17 fitness genes showed significant reductions (up to 73%) for eggs/g root in the T3 and T4 homozygous transgenic lines. Targeted mRNAs of SCN eggs collected from the transgenic soybean lines were efficiently down-regulated, as confirmed by quantitative RT-PCR. Based on the small RNA-seq data and bioassays, it is our hypothesis that a threshold of small interfering RNA molecules is required to significantly reduce SCN populations feeding on the host plants. Our results demonstrated that host-derived gene silencing of essential SCN fitness genes could be an effective strategy for enhancing resistance in crop plants.

Genome-wide identification of soybean microRNA responsive to soybean cyst nematodes infection by deep sequencing.

BACKGROUND: The soybean cyst nematode (SCN), Heterodera glycines, is one of the most devastating diseases limiting soybean production worldwide. It is known that small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play important roles in regulating plant growth and development, defense against pathogens, and responses to environmental changes. RESULTS: In order to understand the role of soybean miRNAs during SCN infection, we analyzed 24 small RNA libraries including three biological replicates from two soybean cultivars (SCN susceptible KS4607, and SCN HG Type 7 resistant KS4313N) that were grown under SCN-infested and -noninfested soil at two different time points (SCN feeding establishment and egg production). In total, 537 known and 70 putative novel miRNAs in soybean were identified from a total of 0.3 billion reads (average about 13.5 million reads for each sample) with the programs of Bowtie and miRDeep2 mapper. Differential expression analyses were carried out using edgeR to identify miRNAs involved in the soybean-SCN interaction. Comparative analysis of miRNA profiling indicated a total of 60 miRNAs belonging to 25 families that might be specifically related to cultivar responses to SCN. Quantitative RT-PCR validated similar miRNA interaction patterns as sequencing results. CONCLUSION: These findings suggest that miRNAs are likely to play key roles in soybean response to SCN. The present work could provide a framework for miRNA functional identification and the development of novel approaches for improving soybean SCN resistance in future studies.

Caenorhabditis elegans genomic response to soil bacteria predicts environment-specific genetic effects on life history traits.

With the post-genomic era came a dramatic increase in high-throughput technologies, of which transcriptional profiling by microarrays was one of the most popular. One application of this technology is to identify genes that are differentially expressed in response to different environmental conditions. These experiments are constructed under the assumption that the differentially expressed genes are functionally important in the environment where they are induced. However, whether differential expression is predictive of functional importance has yet to be tested. Here we have addressed this expectation by employing Caenorhabditis elegans as a model for the interaction of native soil nematode taxa and soil bacteria. Using transcriptional profiling, we identified candidate genes regulated in response to different bacteria isolated in association with grassland nematodes or from grassland soils. Many of the regulated candidate genes are predicted to affect metabolism and innate immunity suggesting similar genes could influence nematode community dynamics in natural systems. Using mutations that inactivate 21 of the identified genes, we showed that most contribute to lifespan and/or fitness in a given bacterial environment. Although these bacteria may not be natural food sources for C. elegans, we show that changes in food source, as can occur in environmental disturbance, can have a large effect on gene expression, with important consequences for fitness. Moreover, we used regression analysis to demonstrate that for many genes the degree of differential gene expression between two bacterial environments predicted the magnitude of the effect of the loss of gene function on life history traits in those environments.

Several Grassland Soil Nematode Species Are Insensitive to RNA-Mediated Interference.

Phenotypic analysis of defects caused by RNA mediated interference (RNAi) in Caenorhabditis elegans has proven to be a powerful tool for determining gene function. In this study we investigated the effectiveness of RNAi in four non-model grassland soil nematodes, Oscheius sp FVV-2., Rhabditis sp, Mesorhabditis sp., and Acrobeloides sp. In contrast to reference experiments performed using C. elegans and Caenorhabditis briggsae, feeding bacteria expressing dsRNA and injecting dsRNA into the gonad did not produce the expected RNAi knockdown phenotypes in any of the grassland nematodes. Quantitative reverse-transcribed PCR (qRT-PCR) assays did not detect a statistically significant reduction in the mRNA levels of endogenous genes targeted by RNAi in Oscheius sp., and Mesorhabditis sp. From these studies we conclude that due to low effectiveness and inconsistent reproducibility, RNAi knockdown phenotypes in non-Caenorhabditis nematodes should be interpreted cautiously.

Biotechnological application of functional genomics towards plant-parasitic nematode control.

Plant-parasitic nematodes are primary biotic factors limiting the crop production. Current nematode control strategies include nematicides, crop rotation and resistant cultivars, but each has serious limitations. RNA interference (RNAi) represents a major breakthrough in the application of functional genomics for plant-parasitic nematode control. RNAi-induced suppression of numerous genes essential for nematode development, reproduction or parasitism has been demonstrated, highlighting the considerable potential for using this strategy to control damaging pest populations. In an effort to find more suitable and effective gene targets for silencing, researchers are employing functional genomics methodologies, including genome sequencing and transcriptome profiling. Microarrays have been used for studying the interactions between nematodes and plant roots and to measure both plants and nematodes transcripts. Furthermore, laser capture microdissection has been applied for the precise dissection of nematode feeding sites (syncytia) to allow the study of gene expression specifically in syncytia. In the near future, small RNA sequencing techniques will provide more direct information for elucidating small RNA regulatory mechanisms in plants and specific gene silencing using artificial microRNAs should further improve the potential of targeted gene silencing as a strategy for nematode management.

Host-derived suppression of nematode reproductive and fitness genes decreases fecundity of Heterodera glycines Ichinohe.

To control Heterodera glycines Ichinohe (soybean cyst nematode) in Glycine max (L.) Merr. (soybean), we evaluated the use of producing transgenic soybean seedlings expressing small interfering RNAs (siRNAs) against specific H. glycines genes. Gene fragments of three genes related to nematode reproduction or fitness (Cpn-1, Y25 and Prp-17) were PCR-amplified using specific primers and independently cloned into the pANDA35HK RNAi vector using a Gateway cloning strategy. Soybean roots were transformed with these constructions using a composite plant system. Confirmation of transformation was attained by PCR and Southern blot analysis. Transgene expression was detected using reverse transcription PCR (RT-PCR) and expression of siRNAs was confirmed in transgenic plants using northern blot analysis. Bioassays performed on transgenic composite plants expressing double-stranded RNA fragments of Cpn-1, Y25 and Prp-17 genes resulted in a 95, 81 and 79% reduction for eggs g(-1) root, respectively. Furthermore, we demonstrated a significant reduction in transcript levels of the Y25 and Prp-17 genes of the nematodes feeding on the transgenic roots via real-time RT-PCR whereas the expression of non-target genes were not affected. The results of this study demonstrate that over-expression of RNA interference constructs of nematode reproduction or fitness-related genes can effectively control H. glycines infection with levels of suppression comparable to conventional resistance.

Rapid in planta evaluation of root expressed transgenes in chimeric soybean plants.

Production of stable transgenic plants is one factor that limits rapid evaluation of tissue specific transgene expression. To hasten the assessment of transgenes in planta, we evaluated the use of chimeric soybean seedlings expressing transgenic products in roots. Tap roots from four-day old seedlings (cultivars 'Jack' and KS4704) were excised and hairy roots were induced from hypocotyls via Agrobacterium rhizogenes-mediated transformation. Inoculated hypocotyls were screened on a MS-based medium containing either 200 mg/L kanamycin or 20 mg/L hygromycin. Beta-glucuronidase (GUS) activity assay indicated that highest GUS expression was observed in hypocotyls exposed to a 4-d pre-inoculation time, a neutral pH (7.0) for the co-cultivation medium. A 170-bp of the Fib-1 gene and 292-bp of the Y25C1A.5 gene fragments, both related to nematode reproduction and fitness, were cloned independently into pANDA35HK vector using a Gateway cloning strategy. The resulting RNAi constructs of the genes fragments were transformed into soybean using the chimeric hairy root system and evaluated for its effect on soybean cyst nematode (Heterodera glycines) fecundity. Confirmation of transformation was attained by polymerase chain reaction and Southern-blot analysis, and some potential for suppression of H. glycines reproduction was detected for the two constructs. This method takes on average four weeks to produce chimeric plants ready for transgene analysis.

Host-Derived Artificial MicroRNA as an Alternative Method to Improve Soybean Resistance to Soybean Cyst Nematode.

The soybean cyst nematode (SCN), Heterodera glycines, is one of the most important pests limiting soybean production worldwide. Novel approaches to managing this pest have focused on gene silencing of target nematode sequences using RNA interference (RNAi). With the discovery of endogenous microRNAs as a mode of gene regulation in plants, artificial microRNA (amiRNA) methods have become an alternative method for gene silencing, with the advantage that they can lead to more specific silencing of target genes than traditional RNAi vectors. To explore the application of amiRNAs for improving soybean resistance to SCN, three nematode genes (designated as J15, J20, and J23) were targeted using amiRNA vectors. The transgenic soybean hairy roots, transformed independently with these three amiRNA vectors, showed significant reductions in SCN population densities in bioassays. Expression of the targeted genes within SCN eggs were downregulated in populations feeding on transgenic hairy roots. Our results provide evidence that host-derived amiRNA methods have great potential to improve soybean resistance to SCN. This approach should also limit undesirable phenotypes associated with off-target effects, which is an important consideration for commercialization of transgenic crops.

Long-term nitrogen amendment alters the diversity and assemblage of soil bacterial communities in tallgrass prairie.

Anthropogenic changes are altering the environmental conditions and the biota of ecosystems worldwide. In many temperate grasslands, such as North American tallgrass prairie, these changes include alteration in historically important disturbance regimes (e.g., frequency of fires) and enhanced availability of potentially limiting nutrients, particularly nitrogen. Such anthropogenically-driven changes in the environment are known to elicit substantial changes in plant and consumer communities aboveground, but much less is known about their effects on soil microbial communities. Due to the high diversity of soil microbes and methodological challenges associated with assessing microbial community composition, relatively few studies have addressed specific taxonomic changes underlying microbial community-level responses to different fire regimes or nutrient amendments in tallgrass prairie. We used deep sequencing of the V3 region of the 16S rRNA gene to explore the effects of contrasting fire regimes and nutrient enrichment on soil bacterial communities in a long-term (20 yrs) experiment in native tallgrass prairie in the eastern Central Plains. We focused on responses to nutrient amendments coupled with two extreme fire regimes (annual prescribed spring burning and complete fire exclusion). The dominant bacterial phyla identified were Proteobacteria, Verrucomicrobia, Bacteriodetes, Acidobacteria, Firmicutes, and Actinobacteria and made up 80% of all taxa quantified. Chronic nitrogen enrichment significantly impacted bacterial community diversity and community structure varied according to nitrogen treatment, but not phosphorus enrichment or fire regime. We also found significant responses of individual bacterial groups including Nitrospira and Gammaproteobacteria to long-term nitrogen enrichment. Our results show that soil nitrogen enrichment can significantly alter bacterial community diversity, structure, and individual taxa abundance, which have important implications for both managed and natural grassland ecosystems.

Molecular approach for assessing responses of microbial-feeding nematodes to burning and chronic nitrogen enrichment in a native grassland.

A substantial proportion of the primary productivity in grassland ecosystems is allocated belowground, sustaining an abundant and diverse community of microbes and soil invertebrates. These belowground communities drive many important ecosystem functions and are responsive to a variety of environmental changes. Nematodes, an abundant and diverse component of grassland soil communities, are particularly responsive to altered environmental conditions, such as those associated with reduced fire frequency and nitrogen enrichment, with the most consistent responses displayed by microbial-feeding nematodes. However, much of the available research characterizing nematode responses to environmental change has been carried out at the taxonomic level of family or by broad trophic categories (e.g. fungivores, bacterivores). The extent to which differential responses to environmental change occurs at the genus level or below is unclear. Therefore, the objective of this study was to use molecular methods to quantify the response of microbial-feeding nematodes, at the lowest levels of taxonomic resolution, to nitrogen enrichment and changes in fire frequency. Using sequencing and quantitative polymerase chain reaction (PCR) probes for the 18S ribosomal RNA gene and the ITS1 region, we identified 19 microbial-feeding nematode taxa across four families. When nematodes were sampled across treatments, we found that some nematode taxa within a family responded similarly to nitrogen and burning treatments, while other taxa within the same family respond quite differently. Additionally, although nematodes from different families on average responded differently to nitrogen enrichment and burning, similar responses were seen in nematode taxa that span three taxonomic families. Thus, if nematodes are to be used as indicators of environmental change, care should be taken to assess the response at the lowest taxonomic level possible.