rna-blot-analysis-for-detection-quantification-plant

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...

Tata Institute of Fundamental Research

Micro (mi)RNAs are important regulators of plant development. Across plant lineages, Dicer-like 1 (DCL1) proteins process long ds-like structures to produce micro (mi) RNA duplexes in a stepwise manner. These miRNAs are incorporated into Argonaute (AGO) proteins and influence expression of RNAs that have sequence complementarity with miRNAs. Expression levels of AGOs are greatly regulated by plants in order to minimize unwarranted perturbations using miRNAs to target mRNAs coding for AGOs. AGOs may also have high promoter specificity-sometimes expression of AGO can be limited to just a few cells in a plant. Viral pathogens utilize various means to counter antiviral roles of AGOs including hijacking the host encoded miRNAs to target AGOs. Two host encoded miRNAs namely miR168 and miR403 that target AGOs have been described in the model plant Arabidopsis and such a mechanism is thought to be well conserved across plants because AGO sequences are well conserved.

Diversity, expression and mRNA targeting abilities of Argonaute-targeting miRNAs among selected vascular plants.

Methylglyoxal (MG) is a key signaling molecule resulting from glycolysis and other metabolic pathways. During abiotic stress, MG levels accumulate to toxic levels in affected cells. However, MG is routinely detoxified through the action of DJ1/PARK7/Hsp31 proteins that are highly conserved across kingdoms and mutations in such genes are associated with neurodegenerative diseases. Here, we report for the first time that, similar to abiotic stresses, MG levels increase during biotic stresses in plants, likely contributing to enhanced susceptibility to a wide range of stresses. We show that overexpression of yeast Heat shock protein 31 (Hsp31), a DJ-1 homolog with robust MG detoxifying capabilities, confers dual biotic and abiotic stress tolerance in model plant Nicotiana tabacum. Strikingly, overexpression of Hsp31 in tobacco imparts robust stress tolerance against diverse biotic stress inducers such as viruses, bacteria and fungi, in addition to tolerance against a range of abiotic stress inducers. During stress, Hsp31 was targeted to mitochondria and induced expression of key stress-related genes. These results indicate that Hsp31 is a novel attractive tool to engineer plants against both biotic and abiotic stresses.

Methylglyoxal detoxification by a DJ-1 family protein provides dual abiotic and biotic stress tolerance in transgenic plants.

Begomoviruses (family Geminiviridae) cause important diseases in many crops. In addition, many wild plants are reservoirs of begomoviruses, which are a potential menace for nearby crops. A novel begomovirus was isolated from a weed of the species Synedrella nodiflora (Compositae) exhibiting yellow vein symptoms in South India. This virus had a typical monopartite Old World begomovirus genome and was accompanied by a betasatellite. Sequence comparison revealed that this virus represents a new species in the genus Begomovirus. Recombination analysis showed that the novel begomovirus originated through recombination between the begomoviruses ageratum yellow vein Sri Lanka virus and tomato leaf curl Sri Lanka virus.

Molecular characterization of a new begomovirus infecting Synedrella nodiflora in South India.

Domestication of rice () included conversion of perennial wild species with few seeds to short plants that produced abundant seeds. Most domestication-associated changes were due to variations in transcription factors and other key proteins such as enzymes. Here, we show that multiple yield-related traits associated with  rice domestication are linked to micro (mi) RNA-mediated regulation. Analysis of small (s) RNA data sets from cultivated indica rice lines, a few landraces, and two wild relatives of rice revealed the presence of abundant 22-nucleotide (nt) reads in wild relatives that mapped to miR397 precursors. miR397 was expressed at very high levels in wild relatives and at negligible levels in high-yielding cultivated lines. In its genera-specific form of 22-nt, miR397 targeted mRNAs encoding laccases that decayed and induced robust secondary cascade silencing in wild species that required RNA-dependent RNA polymerase 6. In wild species of rice, reduced expression of laccases resulted in low lignification. As expected, overexpression of miR397 induced de-domestication phenotypes. At least 26 uncharacterized QTLs previously implicated in rice yield overlapped with laccases and miR397 genes. These results suggest that miRNAs contribute to rice domestication-associated phenotypes.

Major Domestication-Related Phenotypes in  Rice Are Due to Loss of miRNA-Mediated Laccase Silencing.

Domestication of rice involved incorporation of specific yield-related changes in wild species of rice. This agricultural process has been of significant interest for plant biologists. The recent advance in genomics has provided new tools to investigate the genetic basis and consequences of domestication. Several genes involved in domestication and diversification process have been characterized, and as expected, this list is over-represented by transcription factors and their cofactors. Most often the modification orchestrated expression levels of genes such as those coding for transcription factors. It has been proposed that transcriptional regulators and their regulation is likely a major theme controlling morphological differences between crops and their progenitors. However, recent data indicate that single amino acid changes in genes coding for key proteins as well as epigenetic and small RNA-mediated pathways also contributed towards domesticationassociated phenotypes.

New clues into the mechanisms of rice domestication.

In this manuscript, we show that Oryza sativa indica Argonaute protein AGO18 is required for male gametophyte development likely to through a small RNA-mediated mechanism. Monocots have evolved unique gene silencing pathways due to the presence of unique members of Dicer-like and Argonaute (AGO) family members. Among the monocot AGO homologs, AGO18 occupies a unique position. Previous reports have implicated this protein in viral resistance as well as in gametogenesis, likely through its competition with AGO1 clade members for micro(mi)RNAs and other small (s)RNAs. Although expression of rice AGO18 in specific stages of male gametogenesis has been documented, its major functions in plant development remain poorly understood. Here, we show that Oryza sativa indica AGO18 is involved in male gametophyte development. Knockdown (KD) of AGO18 in transgenic rice lines resulted in stunted plants that are male sterile, whereas their carpels were functional. Transcriptome analysis revealed downregulation of several pollen development-associated genes in KD lines. sRNA sequencing in vegetative and reproductive tissues of KD lines indicated reduction of miRNAs and phased secondary sRNAs implicated in male gametophyte development. Our results indicate a distinct role for rice AGO18 in male fertility.

P. V. Shivaprasad

National Centre for Biological Sciences

Tata Institute of Fundamental Research

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

P. V. Shivaprasad

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Tata Institute of Fundamental Research

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

National Centre for Biological Sciences