Legumes and many nonleguminous plants enter symbiotic interactions with microbes, and it is poorly understood how host plants respond to promote beneficial, symbiotic microbial interactions while suppressing those that are deleterious or pathogenic. Trans-acting siRNAs (tasiRNAs) negatively regulate target transcripts and are characterized by siRNAs spaced in 21-nucleotide (nt) "phased" intervals, a pattern formed by DICER-LIKE 4 (DCL4) processing. A search for phased siRNAs (phasiRNAs) found at least 114 Medicago loci, the majority of which were defense-related NB-LRR-encoding genes. We identified three highly abundant 22-nt microRNA (miRNA) families that target conserved domains in these NB-LRRs and trigger the production of trans-acting siRNAs. High levels of small RNAs were matched to >60% of all ∼540 encoded Medicago NB-LRRs; in the potato, a model for mycorrhizal interactions, phasiRNAs were also produced from NB-LRRs. DCL2 and SGS3 transcripts were also cleaved by these 22-nt miRNAs, generating phasiRNAs, suggesting synchronization between silencing and pathogen defense pathways. In addition, a new example of apparent "two-hit" phasiRNA processing was identified. Our data reveal complex tasiRNA-based regulation of NB-LRRs that potentially evolved to facilitate symbiotic interactions and demonstrate miRNAs as master regulators of a large gene family via the targeting of highly conserved, protein-coding motifs, a new paradigm for miRNA function.

microRNAs as Master Regulators of the Plant NB-LRR Defense Gene Family via the Production of Phased, Trans-acting siRNAs

microRNAs (miRNAs) are a class of negative regulators that take part in many processes such as growth and development, stress responses, and metabolism in plants. Recently, miRNAs were shown to function in plant nutrient metabolism. Moreover, several miRNAs were identified in the response to nitrogen (N) deficiency. To investigate the functions of other miRNAs in N deficiency, deep sequencing technology was used to detect the expression of small RNAs under N-sufficient and -deficient conditions. The results showed that members from the same miRNA families displayed differential expression in response to N deficiency. Upon N starvation, the expression of miR169, miR171, miR395, miR397, miR398, miR399, miR408, miR827, and miR857 was repressed, whereas those of miR160, miR780, miR826, miR842, and miR846 were induced. miR826, a newly identified N-starvation-induced miRNA, was found to target the AOP2 gene. Among these N-starvation-responsive miRNAs, several were involved in cross-talk among responses to different nutrient (N, P, S, Cu) deficiencies. miR160, miR167, and miR171 could be responsible for the development of Arabidopsis root systems under N-starvation conditions. In addition, twenty novel miRNAs were identified and nine of them were significantly responsive to N-starvation. This study represents comprehensive expression profiling of N-starvation-responsive miRNAs and advances our understanding of the regulation of N homeostasis mediated by miRNAs.

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Identification of Nitrogen Starvation-Responsive MicroRNAs in Arabidopsis thaliana

The phenomenon of RNA interference (RNAi) which involves sequence specific gene regulation by small non-coding RNAs ie small interfering RNA (siRNA) and micro RNA (miRNA) has emerged as one of most powerful approaches for crop improvement RNAi based on siRNA is one of the widely used tools of reverse genetics which aid in revealing gene functions in many species This technology has been extensively applied to alter the gene expression in plants with an aim to achieve desirable traits RNAi has been used for enhancing the crop yield and productivity by manipulating the gene involved in biomass, grain yield and enhanced shelf life of fruits & vegetables It has also been applied for developing resistance against various biotic (bacteria, fungi, viruses, nematodes, insects) and abiotic stresses (drought, salinity, cold etc) Nutritional improvements of crops have also been achieved by enriching the crops with essential amino acids, fatty acids, antioxidants and other nutrients beneficial for human health or by reducing allergens or anti-nutrients Micro RNAs are key regulators of important plant processes like growth, development and response to various stresses In spite of similarity in size (20-24nt), miRNA differ from siRNA in precursor structures, pathway of biogenesis, and modes of action This review also highlights the miRNA based genetic modification technology where various miRNAs/artificial miRNAs and their targets can be utilized for improving several desirable plant traits Micro RNA based strategies are much efficient than siRNA-based RNAi strategies due to its specificity and less undesirable off target effects As per the FDA guidelines, small RNA based transgenics are much safer for consumption than those over expressing proteins This review thereby summarizes the emerging advances and achievement in the field of small RNAs and its application for crop improvement

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Small RNAs in plants: recent development and application for crop improvement.

MicroRNAs (miRNAs) are an extensive class of newly discovered endogenous small RNAs, which negatively regulate gene expression at the post-transcription levels. As the application of next-generation deep sequencing and advanced bioinformatics, the miRNA-related study has been expended to non-model plant species and the number of identified miRNAs has dramatically increased in the past years. miRNAs play a critical role in almost all biological and metabolic processes, and provide a unique strategy for plant improvement. Here, we first briefly review the discovery, history, and biogenesis of miRNAs, then focus more on the application of miRNAs on plant breeding and the future directions. Increased plant biomass through controlling plant development and phase change has been one achievement for miRNA-based biotechnology; plant tolerance to abiotic and biotic stress was also significantly enhanced by regulating the expression of an individual miRNA. Both endogenous and artificial miRNAs may serve as important tools for plant improvement.

MicroRNA-based biotechnology for plant improvement.

Although generally unnoticed, nearly all crop plants have one or more species of nematodes that feed on their roots, frequently causing tremendous yield losses. The group of sedentary nematodes, which are among the most damaging plant-parasitic nematodes, cause the formation of special organs called nematode feeding sites (NFS) in the root tissue. In this review we discuss key metabolic and cellular changes correlated with NFS development, and similarities and discrepancies between different types of NFS are highlighted.

Nematode feeding sites: unique organs in plant roots

Soybean cyst nematode (SCN), Heterodera glycines, is the most devastating pathogen of soybean worldwide. MicroRNAs (miRNAs) are a class of small, non-coding RNAs that are known to play important role in plant stress response. However, there are few reports profiling the miRNA expression patterns during pathogen stress. We sequenced four small RNA libraries from two soybean cultivar (Hairbin xiaoheidou, SCN race 3 resistant, Liaodou 10, SCN race 3 susceptible) that grown under un-inoculated and SCN-inoculated soil. Small RNAs were mapped to soybean genome sequence, 364 known soybean miRNA genes were identified in total. In addition, 21 potential miRNA candidates were identified. Comparative analysis of miRNA profiling indicated 101 miRNAs belong to 40 families were SCN-responsive. We also found 20 miRNAs with different express pattern even between two cultivars of the same species. These findings suggest that miRNA paly important role in soybean response to SCN and have important implications for further identification of miRNAs under pathogen stress.

/pdf/identification-of-soybean-micrornas-involved-in-soybean-cyst-4sce1xsymo.pdf

Identification of Soybean MicroRNAs Involved in Soybean Cyst Nematode Infection by Deep Sequencing

To gain insight into the changes in the transcriptome of soybean roots during soybean cyst nematode (SCN) infection, we conducted genome-wide gene expression profiling using serial analysis of gene expression (SAGE) combined with Solexa sequencing. More than 3 million tags were generated from the SCN-infected and uninfected roots, and 366941 and 314591 clean UniTags were obtained from SCN-infected and uninfected samples, respectively. In the SCN-infected sample, 48249 UniTags represented 18114 reference genes. In the uninfected control, 46290 UniTags represented 19323 reference genes. Comparison of tag frequencies identified 1405 genes that were expressed at greater levels in SCN-infected roots than in uninfected roots, and 1191 genes that were expressed at lower levels. Quantitative real-time PCR analyses confirmed the changes in mRNA levels observed in our sequencing analyses. A comparable number of genes were up- and down-regulated in response to nematode infection, indicating that down-regulation of some genes might be essential in the plant response to nematodes. Our SAGE results showed significant changes in expression of many unreported genes involved in nematode infection. Approximately 7% of tags mapped to the antisense strand of genes, indicating widespread antisense transcription.

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Comparative profiling of the transcriptional response to soybean cyst nematode infection of soybean roots by deep sequencing

Cordyceps sinensis (CS) is well known as an ancient Chinese herb. It is used to expand bronchial smooth muscles, inhibit tumor growth and decrease blood pressure. Cordyceps sinensis is composed of two parts. One is the dead larvae body; the other is the stroma like withered grass. Up to now, few genome database articles about Cordyceps sinensis have been reported. In this study, two cDNA libraries were constructed using the worm part and grass part respectively for the first time. 12192 and 15456 clones from the worm-part (CSCA) and grass-part (CSBA) library were respectively picked. Sequences derived from CSCA were clustered into 1333 contigs and 2469 singlets, while those from CSBA were clustered into 1297 contigs and 2875 singlets. These ESTs include sequences representing a significant portion of proteins encoding genes in cell signalling, metabolism, information storage and processing. Some enzymes encoding genes were also found and linked with CS’s physiology such as proteases, peptidases, lipases and chitinase. Pairwise comparison between the two cDNA libraries was also studied. Some ESTs were found only in CSCA and some only in CSBA. Finally, a comparative genomics research was performed with Fusarium graminearum, Aspergillus nidulans, Neurospora crassa and Saccharomyces bayanus. The results indicate that the fungus’ genes maybe have complicated variation at the nucleic acid level, but the proteins translated are still conservative.

ShaoPeng Zhang

Papers

Identification of Soybean MicroRNAs Involved in Soybean Cyst Nematode Infection by Deep Sequencing

Comparative profiling of the transcriptional response to soybean cyst nematode infection of soybean roots by deep sequencing

Genome research profile of two Cordyceps sinensis cDNA libraries