Elucidation of the MicroRNA Transcriptome in Western Corn Rootworm Reveals Its Dynamic and Evolutionary Complexity.

Diabrotica virgifera virgifera (western corn rootworm, WCR) is one of the most destructive agricultural insect pests in North America. It is highly adaptive to environmental stimuli and crop protection technologies. However, little is known about the underlying genetic basis of WCR behavior and adaptation. More specifically, the involvement of small RNAs (sRNAs), especially microRNAs (miRNAs), a class of endogenous small non-coding RNAs that regulate various biological processes, has not been examined, and the datasets of putative sRNA sequences have not previously been generated for WCR. To achieve a comprehensive collection of sRNA transcriptomes in WCR, we constructed, sequenced, and analyzed sRNA libraries from different life stages of WCR and northern corn rootworm (NCR), and identified 101 conserved precursor miRNAs (pre-miRNAs) in WCR and other Arthropoda. We also identified 277 corn rootworm specific pre-miRNAs. Systematic analyses of sRNA populations in WCR revealed that its sRNA transcriptome, which includes PIWI-interacting RNAs (piRNAs) and miRNAs, undergoes a dynamic change throughout insect development. Phylogenetic analysis of miRNA datasets from model species reveals that a large pool of species-specific miRNAs exists in corn rootworm; these are potentially evolutionarily transient. Comparisons of WCR miRNA clusters to other insect species highlight conserved miRNA-regulated processes that are common to insects. Parallel Analysis of RNA Ends (PARE) also uncovered potential miRNA-guided cleavage sites in WCR. Overall, this study provides a new resource for studying the sRNA transcriptome and miRNA-mediated gene regulation in WCR and other Coleopteran insects.

Plin-amiR, a pre-microRNA-based technology for controlling herbivorous insect pests.

The cotton bollworm, Helicoverpa armigera, is a major insect pest for a wide range of agricultural crops. It causes significant yield loss through feeding damage and by increasing the crop's vulnerability to bacterial and fungal infections. Although expression of Bacillus thuringiensis (Bt) toxins in transgenic crops has been very successful in protecting against insect pests, including H. armigera, field-evolved resistance has occurred in multiple species. To manage resistant populations, new protection strategies must be continuously developed. Trans-kingdom RNA interference (TK-RNAi) is a promising method for controlling herbivorous pests. TK-RNAi is based on delivering dsRNA or hairpin RNA containing essential insect gene sequences to the feeding insect. The ingested molecules are processed by the insect's RNAi machinery and guide it to silence the target genes. Recently, TK-RNAi delivery has been enhanced by expressing the ds- or hpRNAs in the chloroplast. This compartmentalizes the duplexed RNA away from the plant's RNAi machinery, ensuring that it is delivered in an unprocessed form to the insect. Here, we report another alternative approach for delivering precursor anti-insect RNA in plants. Insect pre-microRNA (pre-miR) transcripts were modified to contain artificial microRNAs (amiRs), targeting insect genes, and expressed in transgenic Nicotiana benthamiana plants. These modified pre-miRs remained largely unprocessed in the plants, and H. armigera feeding on leaves from these plants had increased mortality, developmental abnormalities and delayed growth rates. This shows that plant-expressed insect pre-amiRs (plin-amiRs) are a new strategy of protecting plants against herbivorous insects.

The tomato plastidic fructokinase SlFRK3 plays a role in xylem development.

Plants have two kinds of fructokinases (FRKs) that catalyze the key step of fructose phosphorylation, cytosolic and plastidic. The major cytosolic tomato FRK, SlFRK2, is essential for the development of xylem vessels. In order to study the role of SlFRK3, which encodes the only plastidic FRK, we generated transgenic tomato (Solanum lycopersicon) plants with RNAi suppression of SlFRK3 as well as plants expressing beta-glucoronidase (GUS) under the SlFRK3 promoter. GUS staining indicated SlFRK3 expression in vascular tissues of the leaves and stems, including cambium, differentiating xylem, young xylem fibers and phloem companion cells. Suppression of SlFRK3 reduced the stem xylem area, stem and root water conductance, and whole-plant transpiration, with minor effects on plant development. However, suppression of SlFRK3 accompanied by partial suppression of SlFRK2 induced significant growth-inhibition effects, including the wilting of mature leaves. Grafting experiments revealed that these growth effects are imposed primarily by the leaves, whose petioles had unlignified, thin-walled xylem fibers with collapsed parenchyma cells around the vessels. A cross between the SlFRK2-antisense and SlFRK3-RNAi lines exhibited similar wilting and anatomical effects, confirming that these effects are the result of the combined suppression of SlFRK3 and SlFRK2. These results demonstrate a role of the plastidic SlFRK3 in xylem development and hydraulic conductance.

Parallel analysis of RNA ends enhances global investigation of microRNAs and target RNAs of Brachypodium distachyon.

BACKGROUND: The wild grass Brachypodium distachyon has emerged as a model system for temperate grasses and biofuel plants. However, the global analysis of miRNAs, molecules known to be key for eukaryotic gene regulation, has been limited in B. distachyon to studies examining a few samples or that rely on computational predictions. Similarly an in-depth global analysis of miRNA-mediated target cleavage using parallel analysis of RNA ends (PARE) data is lacking in B. distachyon. RESULTS: B. distachyon small RNAs were cloned and deeply sequenced from 17 libraries that represent different tissues and stresses. Using a computational pipeline, we identified 116 miRNAs including not only conserved miRNAs that have not been reported in B. distachyon, but also non-conserved miRNAs that were not found in other plants. To investigate miRNA-mediated cleavage function, four PARE libraries were constructed from key tissues and sequenced to a total depth of approximately 70 million sequences. The roughly 5 million distinct genome-matched sequences that resulted represent an extensive dataset for analyzing small RNA-guided cleavage events. Analysis of the PARE and miRNA data provided experimental evidence for miRNA-mediated cleavage of 264 sites in predicted miRNA targets. In addition, PARE analysis revealed that differentially expressed miRNAs in the same family guide specific target RNA cleavage in a correspondingly tissue-preferential manner. CONCLUSIONS: B. distachyon miRNAs and target RNAs were experimentally identified and analyzed. Knowledge gained from this study should provide insights into the roles of miRNAs and the regulation of their targets in B. distachyon and related plants.

A microRNA of infectious laryngotracheitis virus can downregulate and direct cleavage of ICP4 mRNA.

Viral microRNAs regulate gene expression using either translational repression or mRNA cleavage and decay. Two microRNAs from infectious laryngotracheitis virus (ILTV), iltv-miR-I5 and iltv-miR-I6, map antisense to the ICP4 gene. Post-transcriptional repression by these microRNAs was tested against a portion of the ICP4 coding sequence cloned downstream of firefly luciferase. Luciferase activity was downregulated by approximately 60% with the iltv-miR-I5 mimic. Addition of an iltv-miR-I5 antagomiR or mutagenesis of the target seed sequence alleviated this effect. The iltv-miR-I5 mimic, when co-transfected with a plasmid expressing ICP4, reduced ICP4 transcript levels by approximately 50%, and inhibition was relieved by an iltv-miR-I5 antagomiR. In infected cells, iltv-miR-I5 mediated cleavage at the canonical site, as indicated by modified RACE analysis. Thus, in this system, iltv-miR-I5 decreased ILTV ICP4 mRNA levels via transcript cleavage and degradation. Downregulation of ICP4 could impact the balance between the lytic and latent states of the virus in vivo.

Abiotic stress-associated miRNAs: detection and functional analysis.

MicroRNAs (miRNAs) are small regulatory noncoding RNAs varying in length between 20 and 24 nucleotides. They play a key role during plant development by negatively regulating gene expression at the posttranscriptional level. Moreover, recent studies reported several miRNAs associated with abiotic stress responses. Small RNA cloning and high-throughput deep sequencing methods provide expression profiles of not only known miRNAs, but also novel miRNAs. In this chapter, we describe the methods used to identify and characterize abiotic stress-associated miRNAs and their target genes.

LeFRK2 is required for phloem and xylem differentiation and the transport of both sugar and water.

It has been suggested that LeFRK2, the major fructose-phosphorylating enzyme in tomato plants, may be required for stem xylem development. Yet, we do not know if this enzyme affects the development of individual vessels, whether it affects water conductance, or whether it affects phloem development and sugar transport. Here, we show that suppression of LeFRK2 results in a significant reduction in the size of vascular cells and slows fiber maturation. The vessels in stems of LeFRK2-antisense plants are narrower than in WT plants and have thinner secondary cell walls. Although the cambium produces rounded secondary vessels, these vessels become deformed during the early stages of xylem maturation. Water conductance is then reduced in stems, roots, and leaves, suggesting that LeFRK2 influences xylem development throughout the entire vascular system. Interestingly, the build-up of positive xylem pressure under static (no-flow) conditions was also decreased. Suppression of LeFRK2 reduced the length and width of the sieve elements, as well as callose deposition. To examine the effect of LeFRK2 suppression on phloem transport, we created triple-grafted plants in which a portion of the wild-type stem was replaced with an antisense interstcok, and compared the contents of the transported sugar, sucrose, in the different portions of these stems. Sucrose contents above and within the LeFRK2-antisense interstock were significantly higher than those below the graft. These results show that the antisense interstock restricted the downward movement of sucrose, suggesting that LeFRK2 is required for both phloem and xylem development.

Construction of Parallel Analysis of RNA Ends (PARE) libraries for the study of cleaved miRNA targets and the RNA degradome.

We have developed a novel approach called parallel analysis of RNA ends (PARE) for high-throughput identification of microRNA (miRNA) targets and diverse applications for the study of the RNA degradome. The method described here comprises a modified 5'-rapid amplification of cDNA ends, deep sequencing of 3' cleavage products of mRNA and bioinformatic analysis. Following RNA extraction and isolation of polyadenylated RNA, a 5'-RNA adapter that includes an MmeI recognition site is ligated to 5'-monophosphorylated products, which contain mRNA fragments generated through miRNA-induced cleavage. The ligated products are reverse-transcribed, slightly amplified and cleaved with MmeI. The 5' equally-sized fragments are gel-selected, ligated to a 3' double-stranded DNA adapter and PCR-amplified. Following gel purification, the products are subjected to deep sequencing. The data are then matched to cDNAs and analyzed through bioinformatics filters. We describe the high-throughput protocol in detail and indicate alternative uses for PARE. The procedure presented here can be accomplished in 6-7 d.

Global identification of microRNA-target RNA pairs by parallel analysis of RNA ends.

MicroRNAs (miRNAs) are important regulatory molecules in most eukaryotes and identification of their target mRNAs is essential for their functional analysis. Whereas conventional methods rely on computational prediction and subsequent experimental validation of target RNAs, we directly sequenced >28,000,000 signatures from the 5' ends of polyadenylated products of miRNA-mediated mRNA decay, isolated from inflorescence tissue of Arabidopsis thaliana, to discover novel miRNA-target RNA pairs. Within the set of approximately 27,000 transcripts included in the 8,000,000 nonredundant signatures, several previously predicted but nonvalidated targets of miRNAs were found. Like validated targets, most showed a single abundant signature at the miRNA cleavage site, particularly in libraries from a mutant deficient in the 5'-to-3' exonuclease AtXRN4. Although miRNAs in Arabidopsis have been extensively investigated, working in reverse from the cleaved targets resulted in the identification and validation of novel miRNAs. This versatile approach will affect the study of other aspects of RNA processing beyond miRNA-target RNA pairs.

Suppression of fructokinase encoded by LeFRK2 in tomato stem inhibits growth and causes wilting of young leaves.

Fructokinases catalyze the key step of fructose phosphorylation in plants. LeFRK2, the major fructokinase-encoding gene in tomato plants, is abundantly expressed in roots, stems, and fruits. To analyze the role of LeFRK2 in plant development, we analyzed transgenic tomato plants with sense and antisense expression of StFRK, the potato homolog of LeFRK2. Increased fructokinase activity had no effect. However, plants in which LeFRK2 was specifically suppressed, either via antisense suppression or via co-suppression, exhibited growth inhibition and wilting of young leaves at daytime. Grafting experiments indicated that a stem interstock of antisense plants was sufficient to inhibit growth and cause leaf wilting. Stem secondary xylem exhibited particular suppression of LeFRK2 and the area of active xylem, estimated by eosin uptake, was significantly smaller in antisense stem compared to that of wild-type plants. These results suggest that LeFRK2 might be required for proper development of xylem that affected growth and wilting.