Botrytis cinerea, the causative agent of gray mold disease, is an aggressive fungal pathogen that infects more than 200 plant species. Here, we show that some B. cinerea small RNAs (Bc-sRNAs) can silence Arabidopsis and tomato genes involved in immunity. These Bc-sRNAs hijack the host RNA interference (RNAi) machinery by binding to Arabidopsis Argonaute 1 (AGO1) and selectively silencing host immunity genes. The Arabidopsis ago1 mutant exhibits reduced susceptibility to B. cinerea, and the B. cinerea dcl1 dcl2 double mutant that can no longer produce these Bc-sRNAs displays reduced pathogenicity on Arabidopsis and tomato. Thus, this fungal pathogen transfers “virulent” sRNA effectors into host plant cells to suppress host immunity and achieve infection, which demonstrates a naturally occurring cross-kingdom RNAi as an advanced virulence mechanism.

/pdf/fungal-small-rnas-suppress-plant-immunity-by-hijacking-host-4eq92r0ihp.pdf

Fungal Small RNAs Suppress Plant Immunity by Hijacking Host RNA Interference Pathways

Coordinate Regulation of FOXO1 by miR-27a, miR-96, and miR-182 in Breast Cancer Cells

The timing of the induction of flowering determines to a large extent the reproductive success of plants. Plants integrate diverse environmental and endogenous signals to ensure the timely transition from vegetative growth to flowering. Carbohydrates are thought to play a crucial role in the regulation of flowering, and trehalose-6-phosphate (T6P) has been suggested to function as a proxy for carbohydrate status in plants. The loss of TREHALOSE-6-PHOSPHATE SYNTHASE 1 (TPS1) causes Arabidopsis thaliana to flower extremely late, even under otherwise inductive environmental conditions. This suggests that TPS1 is required for the timely initiation of flowering. We show that the T6P pathway affects flowering both in the leaves and at the shoot meristem, and integrate TPS1 into the existing genetic framework of flowering-time control.

http://science.sciencemag.org/content/sci/339/6120/704.full.pdf

Regulation of Flowering by Trehalose-6-Phosphate Signaling in Arabidopsis thaliana

Transcriptional Control of Gene Expression by MicroRNAs

https://web.nmsu.edu/~plantgen/Web_resources_files/Rashmi%20Tiwari.pdf

Differential expression of miRNAs in response to salt stress in maize roots

MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression.

/pdf/protocol-a-highly-sensitive-rt-pcr-method-for-detection-and-26ru91n1ic.pdf

Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs

MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 l of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression.

Protocol : A highly sensitive RT-PCR method for detection and quantification of microRNAs

Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs). The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified. This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.

/pdf/analysis-of-expressed-sequence-tags-from-actinidia-486pbpgiuy.pdf

Analysis of expressed sequence tags from Actinidia: Applications of a cross species EST database for gene discovery in the areas of flavor, health, color and ripening

MicroRNAs (miRNAs) are naturally occurring, highly conserved families of transcripts (18–25 nt in length) that are processed from larger hairpin precursors. Both plant and animal genomes encode miRNAs, and to date, there are over 4,000 mature miRNA transcripts annotated. Although miRNAs represent a relatively abundant class of transcripts, their expression levels vary greatly among species and tissues. Less abundant miRNAs routinely fail detection using technologies such as cloning, northern hybridization and microarray analysis. Here, we present a real-time quantification method for accurate, sensitive, and economical detection of plant miRNAs based upon the stem-loop RT primer approach using an miRNA-specific primer coupled with a stem-loop specific Universal ProbeLibrary (UPL) probe. This approach represents a highly specific, sensitive, and easy-to-apply system to detect miRNAs.

Marion Wood

Papers

Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs

Protocol : A highly sensitive RT-PCR method for detection and quantification of microRNAs

Analysis of expressed sequence tags from Actinidia: Applications of a cross species EST database for gene discovery in the areas of flavor, health, color and ripening

Real-Time PCR Quantification of Plant miRNAs Using Universal ProbeLibrary Technology