@article {161132, title = {Immunofluorescence Staining and Microscopic Imaging of Plant Nuclei for Epigenetic Modifications.}, journal = {Methods Mol Biol}, volume = {2988}, year = {2026}, month = {2026}, pages = {83-92}, abstract = {

Histone posttranslational modifications (PTMs) and DNA methylation are the predominant epigenetic modifications on the chromatin that regulate gene expression. These modifications can be spatially resolved using microscopic examination of the nuclei with the help of commercially available antibodies. Here, we describe a detailed method to obtain intact nuclei from plant tissues and reproducibly immunostain the nuclei for specific chromatin marks and microscopic examination. This method can be readily extended to multiple plant species as the antibodies are raised against conserved epigenetic marks.

}, keywords = {Cell Nucleus, Chromatin, DNA Methylation, Epigenesis, Genetic, Epigenomics, Fluorescent Antibody Technique, Histones, Microscopy, Fluorescence, Plants, Protein Processing, Post-Translational}, issn = {1940-6029}, doi = {10.1007/978-1-0716-4981-7_8}, author = {Gandhivel, Vivek Hari-Sundar and Raju, Steffi and Shivaprasad, P V} } @article {161131, title = {Targeted Bisulfite Sequencing and DNA Methylation Analysis in Rice Endosperm and Other Tissues.}, journal = {Methods Mol Biol}, volume = {2988}, year = {2026}, month = {2026}, pages = {71-82}, abstract = {

DNA methylation is a major epigenetic mark on chromatin. Tissue-specific DNA methylation in the regulatory regions of key genes modulates their tissue-specific expression. To study DNA methylation levels at specific sequence contexts across tissues or during various environmental conditions, whole-genome DNA methylation analysis is widely used. However, whole-genome methylation analysis is an expensive, time-consuming technique and requires specific skills. To understand the role of DNA methylation in a small number of candidate loci, targeted bisulfite (BS) PCR-sequencing analysis is an attractive option. Here, we describe a detailed method to understand DNA methylation by targeted-BS PCR analysis using rice endosperm tissues. This method can be readily extended to multiple monocots, across various tissues and various stress conditions.

}, keywords = {DNA Methylation, DNA, Plant, Endosperm, Epigenesis, Genetic, Genome, Plant, High-Throughput Nucleotide Sequencing, Oryza, Polymerase Chain Reaction, Sequence Analysis, DNA, Sulfites}, issn = {1940-6029}, doi = {10.1007/978-1-0716-4981-7_7}, author = {Pal, Avik Kumar and Gandhivel, Vivek Hari-Sundar and Shivaprasad, P V} } @article {162677, title = {Histone variants: Distinct building blocks of the chromatin acting at the core.}, journal = {Curr Opin Plant Biol}, volume = {89}, year = {2025}, month = {2025 Dec 04}, pages = {102829}, abstract = {

Histone variants alter the core properties of the nucleosomes they decorate and hence constitute a significant epigenetic layer to control cellular processes. Historically, histone variants have been studied using classical genetics to implicate the functions associated with them. However, over the last few years, advanced (epi)genomics and structural investigations have revealed the fine molecular steps involved in histone variant-specific genome regulation. This review outlines the key mechanistic findings that uncovered both structural and functional aspects of plant histone variants in unprecedented resolution. We also highlight the key avenues that might hold potential for future studies, including chromatin engineering using histone variants.

}, issn = {1879-0356}, doi = {10.1016/j.pbi.2025.102829}, author = {Hari-Sundar Gandhivel, Vivek and Shivaprasad, P V} } @article {156921, title = {An Oryza-specific histone H4 variant predisposes H4 lysine 5 acetylation to modulate salt stress responses}, year = {2025}, abstract = {
Paralogous variants of canonical histones guide accessibility to DNA and function as additional layers of genome regulation. Across eukaryotes, the mechanism of action and functional significance of several variants of core histones are well known except those of histone H4. Here we show that a variant of H4 (H4.V) expressing tissue-specifically among Oryza members mediated specific epigenetic changes contributing to salt tolerance. H4.V was incorporated into specific heterochromatic sites, where it blocked the deposition of active histone marks. Stress-dependent redistribution of H4.V enabled the incorporation of acetylated H4 lysine 5 (H4K5ac) in the gene bodies. The misexpression of H4.V led to defects in reproductive development and in mounting salt stress responses. H4.V formed homotypic nucleosomes and mediated these alterations by conferring distinct molecular properties to the nucleosomes, as seen with cryo electron microscopy structures and biochemical assays. These results reveal not only an H4 variant among plants but also a chromatin regulation that might have contributed to the adaptation of semi-aquatic Oryza members.
}, doi = {10.1038/s41477-025-01974-2}, author = {Vivek Hari Sundar Gandhivel and Paula Sotelo-Parrilla and Steffi Raju and Shaileshanand Jha and Anjitha Gireesh and Chitthavalli Y. Harshith and Fabian Gut and Kutti R. Vinothkumar and Fr{\'e}d{\'e}ric Berger and A. Arockia Jeyaprakash and Shivaprasad, P V} } @article {156893, title = {Transcriptional and epigenomic changes in response to PEG-triggered osmotic stress in rapeseed (Brassica napus L.).}, journal = {J Exp Bot}, year = {2025}, month = {2025 Apr 03}, abstract = {

Drought hinders growth, development, and productivity of higher plants. While physiological and molecular background of plant responses to drought has been extensively studied, the role of post-translational modifications of histones or DNA methylation in response to dehydration remains largely elusive. In this study, we deciphered genome-wide changes in transcriptome and histones modifications in response to dehydration in rapeseed (Brassica napus L.). High-throughput transcript profiling (RNA-seq) and chromatin immunoprecipitation followed by sequencing (ChIP-seq) of PEG-treated rapeseed plants revealed genome-scale changes in transcription and histone methylation patterns, specifically in histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 tri-methylated lysine 27 (H3K27me3) sites. We have identified gene sets with altered transcript profiles as well as histone methylation marks in response to osmotic stress. Several proline biosynthesis regulatory genes coding for Delta 1-Pyrroline-5-Carboxylate Synthetases (P5CS) displayed changes in H3K4me3 and/or H3K36me3 enrichment post PEG treatment. Targeted bisulphite sequencing further identified stress-dependent gene body DNA methylation in one of the BnP5CSA gene copies that correlates with its stress-induced activation. By integrating physiological, transcriptional and epigenomic data, our study contributes to a better understanding of the drought response control in crop plants.

}, keywords = {ChIP; DNA methylation; Drought responsive genes; H3K27me3; H3K4me3; Histone methylation; P5CS genes; Proline; Rapeseed; Transcriptome.}, issn = {1460-2431}, doi = {10.1093/jxb/eraf123}, url = {https://pubmed.ncbi.nlm.nih.gov/40178034/}, author = {Prasad, Melvin and Shetty, Prateek and Pal, Avik Kumar and Rig{\'o}, G{\'a}bor and Kant, Kamal and Zsigmond, Laura and Nagy, Istv{\'a}n and Shivaprasad, P V and Szabados, L{\'a}szl{\'o}} } @article {137345, title = {Plant histone variants at the nexus of chromatin readouts, stress and development.}, journal = {Biochim Biophys Acta Gen Subj}, volume = {1868}, year = {2024}, month = {2023 Dec 10}, pages = {130539}, abstract = {

Histones are crucial proteins that are involved in packaging the DNA as condensed chromatin inside the eukaryotic cell nucleus. Rather than being static packaging units, these molecules undergo drastic variations spatially and temporally to facilitate accessibility of DNA to replication, transcription as well as wide range of gene regulatory machineries. In addition, incorporation of paralogous variants of canonical histones in the chromatin is ascribed to specific functions. Given the peculiar requirement of plants to rapidly modulate gene expression levels on account of their sessile nature, histones and their variants serve as additional layers of gene regulation. This review summarizes the mechanisms and implications of distribution, modifications and differential incorporation of histones and their variants across plant genomes, and outlines emerging themes.

}, issn = {1872-8006}, doi = {10.1016/j.bbagen.2023.130539}, author = {G Vivek Hari Sundar and Madhu, Aravind and Archana, A and Shivaprasad, P V} } @article {149375, title = {Upstream regulator of genomic imprinting in rice endosperm is a small RNA-associated chromatin remodeler.}, journal = {Nat Commun}, volume = {15}, year = {2024}, month = {2024 Sep 06}, pages = {7807}, abstract = {

Genomic imprinting is observed in endosperm, a placenta-like seed tissue, where transposable elements (TEs) and repeat-derived small RNAs (sRNAs) mediate epigenetic changes in plants. In imprinting, uniparental gene expression arises due to parent-specific epigenetic marks on one allele but not on the other. The importance of sRNAs and their regulation in endosperm development or in imprinting is poorly understood in crops. Here we show that a previously uncharacterized CLASSY (CLSY)-family chromatin remodeler named OsCLSY3 is essential for rice endosperm development and imprinting, acting as an upstream player in the sRNA pathway. Comparative transcriptome and genetic analysis indicated its endosperm-preferred expression and its likely paternal imprinted nature. These important features are modulated by RNA-directed DNA methylation (RdDM) of tandemly arranged TEs in its promoter. Upon perturbation of OsCLSY3 in transgenic lines, we observe defects in endosperm development and a loss of around 70\% of all sRNAs. Interestingly, well-conserved endosperm-specific sRNAs (siren) that are vital for reproductive fitness in angiosperms are also\ dependent on OsCLSY3. We observed that many imprinted genes and seed development-associated genes are\ under the control of OsCLSY3. These results support an essential role of OsCLSY3 in rice endosperm development and imprinting, and propose similar regulatory strategies involving CLSY3 homologs among other cereals.

}, keywords = {Chromatin Assembly and Disassembly, DNA Methylation, DNA Transposable Elements, Endosperm, Gene Expression Regulation, Plant, Genomic Imprinting, Oryza, Plant Proteins, Plants, Genetically Modified, RNA, Plant, RNA, Small Untranslated}, issn = {2041-1723}, doi = {10.1038/s41467-024-52239-z}, author = {Pal, Avik Kumar and Gandhivel, Vivek Hari-Sundar and Nambiar, Amruta B and Shivaprasad, P V} } @article {128071, title = {Begomoviral beta C1 orchestrates organellar genomic instability to augment viral infection}, journal = {PLANT JOURNAL }, year = {2023}, month = {05/2023}, abstract = {

Chloroplast is the site for transforming light energy to chemical energy. It also acts as a production unit for a variety of defense-related molecules. These defense moieties are necessary to mount a successful counter defense against pathogens, including viruses. Previous studies indicated disruption of chloroplast homeostasis as a basic strategy of Begomovirus for its successful infection leading to the production of vein-clearing, mosaic, and chlorotic symptoms in infected plants. Although begomoviral pathogenicity determinant protein Beta C1 (beta C1) was implicated for pathogenicity, the underlying mechanism was unclear. Here we show that, begomoviral beta C1 directly interferes with the host plastid homeostasis. beta C1 induced DPD1, an organelle-specific nuclease, implicated in nutrient salvage and senescence, as well as modulated the function of a major plastid genome maintainer protein RecA1, to subvert plastid genome. We show that beta C1 was able to physically interact with bacterial RecA and its plant homolog RecA1, resulting in its altered activity. We observed that knocking-down DPD1 during virus infection significantly reduced virus-induced necrosis. These results indicate the presence of a strategy in which a viral protein alters host defense by targeting modulators of chloroplast DNA. We predict that the mechanism identified here might have similarities in other plant-pathogen interactions.

}, doi = {10.1111/tpj.16186}, author = {Nair, Ashwin and Harshith, Chitthavalli Y. Y. and Narjala, Anushree and Shivaprasad, P V} } @article {125201, title = {Indian Himalayan natural Arabidopsis thaliana accessions with abolished miR158 levels exhibit robust miR173-initiated trans-acting cascade silencing.}, journal = {Plant J}, year = {2023}, month = {2023 Mar 08}, abstract = {

Small RNAs such as microRNAs (miRNAs) and small interfering (si)RNAs are short 20-24-nucleotide non-coding RNAs. They are key regulators of gene expression in plants and other organisms. Several 22-nucleotide long miRNAs trigger biogenesis of cascades of trans-acting secondary small interfering RNAs, involved in various developmental and stress responses. Here we show that in Himalayan Arabidopsis thaliana accessions having natural mutations in miR158 locus exhibit robust cascade silencing in pentatricopeptide (PPR)-like locus. Furthermore, we show that these cascade sRNAs trigger tertiary silencing of a gene involved in transpiration and stomatal opening. The natural variants of MIR158 having deletions or insertions led to improper processing of miR158 precursors thereby blocking synthesis of mature miR158. Reduced miR158 led to increased level of its target, a pseudo-pentatricopeptide gene that is otherwise targeted by ta-siRNAs generated by miR173 cascade in other accessions. Using small RNA datasets derived from Indian Himalayan accessions, as well as overexpression and knockout lines of miR158, we show that absence of miR158 led to build up of pseudo-PPR derived tertiary small RNAs. These tertiary sRNAs mediated robust silencing of a gene involved in stomatal closure in Himalayan accessions lacking miR158 expression. We functionally validated the tertiary phasiRNA that targets NHX2 a Na -K /H antiporter protein thereby regulating transpiration and stomatal conductance. Overall, we deciphered a new module of sRNA network, miRNA-TAS-siRNA-pseudogene-tertiary phasiRNA-NHX2 in plant adaptation.

}, issn = {1365-313X}, doi = {10.1111/tpj.16175}, author = {Tripathi, Abhinandan Mani and Singh, Rajneesh and Verma, Ashwani Kumar and Singh, Akanksha and Mishra, Parneeta and Dwivedi, Varun and Narayan, Shiv and Sundar, G Vivek Hari and Shirke, Pramod Arvind and Shivaprasad, P V and Roy, Sribash} } @article {135859, title = {Novel regulators of mitochondrial genome integrity.}, journal = {J Biosci}, volume = {48}, year = {2023}, month = {2023}, abstract = {

Mitochondrial genomes are unusual in that they have very low mutation rates in coding sequences when compared with animals, yeast, and bacteria. It has been proposed that the mitochondrial (mt) DNA damage is preferentially repaired by homologous recombination (HR). In this short review, we summarize and discuss the well-established and recently identified pathways that help in maintaining the integrity of plant mitochondrial genomes.

}, keywords = {Animals, DNA, Mitochondrial, Genome, Mitochondrial, Genome, Plant, Homologous Recombination, Mitochondria, Plants, Saccharomyces cerevisiae}, issn = {0973-7138}, author = {Nair, Ashwin and Shivaprasad, P V} } @article {129504, title = {Plant polymerase IV sensitizes chromatin through histone modifications to preclude spread of silencing into protein-coding domains.}, journal = {Genome Res}, year = {2023}, month = {2023 Jun 05}, abstract = {

Across eukaryotes, gene regulation is manifested via chromatin states roughly distinguished as heterochromatin and euchromatin. The establishment, maintenance, and modulation of the chromatin states is mediated using several factors including chromatin modifiers. However, factors that avoid the intrusion of silencing signals into protein-coding genes are poorly understood. Here we show that a plant specific paralog of RNA polymerase (Pol) II, named Pol IV, is involved in avoidance of facultative heterochromatic marks in protein-coding genes, in addition to its well-established functions in silencing repeats and transposons. In its absence, H3K27 trimethylation (me3) mark intruded the protein-coding genes, more profoundly in genes embedded with repeats. In a subset of genes, spurious transcriptional activity resulted in small(s) RNA production, leading to post-transcriptional gene silencing. We show that such effects are significantly pronounced in rice, a plant with a larger genome with distributed heterochromatin compared with Our results indicate the division of labor among plant-specific polymerases, not just in establishing effective silencing via sRNAs and DNA methylation but also in influencing chromatin boundaries.

}, issn = {1549-5469}, doi = {10.1101/gr.277353.122}, author = {Vivek Hari Sundar G and Swetha, Chenna and Basu, Debjani and Pachamuthu, Kannan and Raju, Steffi and Chakraborty, Tania and Mosher, Rebecca A and Shivaprasad, P V} } @article {112955, title = {Cultivar-specific miRNA-mediated RNA silencing in grapes.}, journal = {Planta}, volume = {256}, year = {2022}, month = {2022 Jun 23}, pages = {17}, abstract = {

MAIN CONCLUSION: In-depth comparative degradome analysis of two domesticated grape cultivars with diverse secondary metabolite accumulation reveals differential miRNA-mediated targeting. Small (s)RNAs such as micro(mi)RNAs and secondary small interfering (si) often work as negative switches of gene expression. In plants, it is well known that miRNAs target and cleave mRNAs that have high sequence complementarity. However, it is not known if there are variations in miRNA-mediated targeting between subspecies and cultivars that have been subjected to vast genetic modifications through breeding and other selections. Here, we have used PAREsnip2 tool for analysis of degradome datasets derived from two contrasting domesticated grape cultivars having varied fruit color, habit and leaf shape. We identified several interesting variations in sRNA targeting using degradome and 5{\textquoteright}RACE analysis between two contrasting grape cultivars that was further correlated using RNA-seq analysis. Several of the differences we identified are associated with secondary metabolic pathways. We propose possible means by which sRNAs might contribute to diversity in secondary metabolites and other development pathways between two domesticated cultivars of grapes.

}, keywords = {Gene Expression Regulation, Plant, MicroRNAs, Plant Breeding, RNA Interference, RNA, Plant, Sequence Analysis, RNA, Vitis}, issn = {1432-2048}, doi = {10.1007/s00425-022-03934-y}, author = {Tirumalai, Varsha and Narjala, Anushree and Swetha, Chenna and G Vivek Hari Sundar and Sujith, T N and Shivaprasad, P V} } @article {105482, title = {Degradome comparison between wild and cultivated rice identifies differential targeting by miRNAs.}, journal = {BMC Genomics}, volume = {23}, year = {2022}, month = {2022 Jan 14}, pages = {53}, abstract = {

BACKGROUND: Small non-coding (s)RNAs are involved in the negative regulation of gene expression, playing critical roles in genome integrity, development and metabolic pathways. Targeting of RNAs by ribonucleoprotein complexes of sRNAs bound to Argonaute (AGO) proteins results in cleaved RNAs having precise and predictable\ 5{\textquoteleft} ends. While tools to study sliced bits of RNAs to confirm the efficiency of sRNA-mediated regulation are available, they are sub-optimal. In this study, we provide an improvised version of a tool with better efficiency to accurately validate sRNA targets.

RESULTS: Here, we improvised the CleaveLand tool to identify additional micro (mi)RNA targets that belong to the same family and also other targets within a specified free energy cut-off. These additional targets were otherwise excluded during the default run. We employed these tools to understand the sRNA targeting efficiency in wild and cultivated rice, sequenced degradome from two rice lines, O. nivara and O. sativa indica Pusa Basmati-1 and analyzed variations in sRNA targeting. Our results indicate the existence of multiple miRNA-mediated targeting differences between domesticated and wild species. For example, Os5NG4 was targeted only in wild rice that might be responsible for the poor secondary wall formation when compared to cultivated rice. We also identified differential mRNA targets of secondary sRNAs that were generated after miRNA-mediated cleavage of primary targets.

CONCLUSIONS: We identified many differentially targeted mRNAs between wild and domesticated rice lines. In addition to providing a step-wise guide to generate and analyze degradome datasets, we showed how domestication altered sRNA-mediated cascade silencing during the evolution of indica rice.

}, keywords = {Argonaute Proteins, Base Sequence, MicroRNAs, Oryza, RNA, Messenger}, issn = {1471-2164}, doi = {10.1186/s12864-021-08288-5}, author = {Swetha, Chenna and Narjala, Anushree and Pandit, Awadhesh and Tirumalai, Varsha and Shivaprasad, P V} } @article {157118, title = {Impact of Phyllosphere on Host Rice Landraces.}, journal = {Microbiol Spectr}, volume = {10}, year = {2022}, month = {2022 Aug 31}, pages = {e0081022}, abstract = {

The genus includes widespread plant-associated bacteria that are abundant in the plant phyllosphere (leaf surfaces), consume plant-secreted methanol, and can produce plant growth-promoting metabolites. However, despite the potential to increase agricultural productivity, their impact on host fitness in the natural environment is relatively poorly understood. Here, we conducted field experiments with three traditionally cultivated rice landraces from northeastern India. We inoculated seedlings with native versus nonnative phyllosphere strains and found significant impacts on plant growth and grain yield. However, these effects were variable. Whereas some isolates were beneficial for their host, others had no impact or were no more beneficial than the bacterial growth medium on its own. Host plant benefits were not consistently associated with colonization and did not have altered phyllosphere microbiome composition, changes in the early expression of plant stress response pathways, or bacterial auxin production. We provide the first demonstration of the benefits of phyllosphere for rice yield under field conditions and highlight the need for further analysis to understand the mechanisms underlying these benefits. Given that the host landrace- relationship was not generalizable, future agricultural applications will require careful testing to identify coevolved host-bacterium pairs that may enhance the productivity of high-value rice varieties. Plants are associated with diverse microbes in nature. Do the microbes increase host plant health, and can they be used for agricultural applications? This is an important question that must be answered in the field rather than in the laboratory or greenhouse. We tested the effects of native, leaf-inhabiting bacteria (genus ) on traditionally cultivated rice varieties in a crop field. We found that inoculation with some bacteria increased rice grain production substantially while a nonnative bacterium reduced plant health. Overall, the effect of bacterial inoculation varied across pairs of rice varieties and their native bacteria. Thus, knowledge of evolved associations between specific bacteria hosted by specific rice varieties is necessary to develop ways to increase the yield of traditional rice landraces and preserve these important sources of cultural and genetic diversity.

}, keywords = {epiphytes; grain yield; host fitness; host-bacterial interaction; phyllosphere; rice landraces.}, issn = {2165-0497}, doi = {10.1128/spectrum.00810-22}, author = {Sanjenbam, Pratibha and Shivaprasad, P V and Agashe, Deepa} } @article {113535, title = {Investigation of Transposon DNA Methylation and Copy Number Variation in Plants Using Southern Hybridisation.}, journal = {Bio Protoc}, volume = {12}, year = {2022}, month = {2022 Jun 05}, pages = {e4432}, abstract = {

Plant genomes are pronouncedly enriched in repeat elements such as transposons. These repeats are epigenetically regulated by DNA methylation. Whole genome high-depth sequencing after bisulfite treatment remains an expensive and laborious method to reliably profile the DNA methylome, especially when considering large genomes such as in crops. Here, we present a simple reproducible Southern hybridisation-based assay to obtain incontrovertible methylation patterns from targeted regions in the rice genome. By employing minor but key modifications, we reliably detected transposon copy number variations over multiple generations. This method can be regarded as a gold standard for validation of epigenetic variations at target loci, and the consequent proliferation of transposons, or segregation in several plant replicates and genotypes. .

}, issn = {2331-8325}, doi = {10.21769/BioProtoc.4432}, author = {Vivek Hari Sundar G and Shivaprasad, P V} } @article {74442, title = {Loss of function of Oryza sativa Argonaute 18 induces male sterility and reduction in phased small RNAs.}, journal = {Plant Reprod}, volume = {33}, year = {2020}, month = {2020 Mar}, pages = {59-73}, abstract = {

KEY MESSAGE: 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.

}, issn = {2194-7961}, doi = {10.1007/s00497-020-00386-w}, author = {Das, Soumita and Swetha, Chenna and Pachamuthu, Kannan and Nair, Ashwin and Shivaprasad, P V} } @article {73639, title = {Phenotypic diversity of Methylobacterium associated with rice landraces in North-East India.}, journal = {PLoS One}, volume = {15}, year = {2020}, month = {2020}, pages = {e0228550}, abstract = {

The ecology and distribution of many bacteria is strongly associated with specific eukaryotic hosts. However, the impact of such host association on bacterial ecology and evolution is not well understood. Bacteria from the genus Methylobacterium consume plant-derived methanol, and are some of the most abundant and widespread plant-associated bacteria. In addition, many of these species impact plant fitness. To determine the ecology and distribution of Methylobacterium in nature, we sampled bacteria from 36 distinct rice landraces, traditionally grown in geographically isolated locations in North-East (NE) India. These landraces have been selected for diverse phenotypic traits by local communities, and we expected that the divergent selection on hosts may have also generated divergence in associated Methylobacterium strains. We determined the ability of 91 distinct rice-associated Methylobacterium isolates to use a panel of carbon sources, finding substantial variability in carbon use profiles. Consistent with our expectation, across spatial scales this phenotypic variation was largely explained by host landrace identity rather than geographical factors or bacterial taxonomy. However, variation in carbon utilisation was not correlated with sugar exudates on leaf surfaces, suggesting that bacterial carbon use profiles do not directly determine bacterial colonization across landraces. Finally, experiments showed that at least some rice landraces gain an early growth advantage from their specific phyllosphere-colonizing Methylobacterium strains. Together, our results suggest that landrace-specific host-microbial relationships may contribute to spatial structure in rice-associated Methylobacterium in a natural ecosystem. In turn, association with specific bacteria may provide new ways to preserve and understand diversity in one of the most important food crops of the world.

}, issn = {1932-6203}, doi = {10.1371/journal.pone.0228550}, author = {Sanjenbam, Pratibha and Buddidathi, Radhika and Venkatesan, Radhika and Shivaprasad, P V and Agashe, Deepa} } @article {83750, title = {Plant-virus-insect tritrophic interactions: insights into the functions of geminivirus virion-sense strand genes.}, journal = {Proc Biol Sci}, volume = {287}, year = {2020}, month = {2020 Oct 14}, pages = {20201846}, abstract = {

The genome of the plant-infecting viruses in the family Geminiviridae is composed of one or two circular single stranded DNA of approximately 2.7-5.2 kb in length. These viruses have emerged as the most devastating pathogen infecting a large number of crops and weeds across the continents. They code for fewer open reading frames (ORFs) through the generation of overlapping transcripts derived from the bidirectional viral promoters. Members of geminiviruses code for up to four ORFs in the virion-sense strand, and their gene expression is regulated by various -elements located at their promoters in the intergenic region. These viral proteins perform multiple functions at every stage of the viral life cycle such as virus transport, insect-mediated virus transmission and suppression of host defence. They impede the host{\textquoteright}s multi-layered antiviral mechanisms including gene silencing (at transcriptional and post-transcriptional levels) and hypersensitive response. This review summarizes the essential role of virion-sense strand encoded proteins in transport of viral genomes within and between plant cells, countering defence in hosts (both plants and the insects), and also in the ubiquitous role in vector-mediated transmission. We highlight the significance of their pro-viral activities in manipulating host-derived innate immune responses and the interaction with whitefly-derived proteins. We also discuss the current knowledge on virus replication and transcription within the insect body.

}, issn = {1471-2954}, doi = {10.1098/rspb.2020.1846}, author = {Vinoth Kumar, R and Shivaprasad, P V} } @article {79807, title = {RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs.}, journal = {J Vis Exp}, year = {2020}, month = {2020 Jul 11}, abstract = {

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 animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.

}, issn = {1940-087X}, doi = {10.3791/61394}, author = {Tirumalai, Varsha and Prasad, Melvin and Shivaprasad, P V} } @article {81664, title = {Stability of Begomoviral pathogenicity determinant βC1 is modulated by mutually antagonistic SUMOylation and SIM interactions.}, journal = {BMC Biol}, volume = {18}, year = {2020}, month = {2020 Aug 31}, pages = {110}, abstract = {

BACKGROUND: To successfully invade new hosts, plant viruses must break host resistance and be competent to move within and between plant cells. As a means, viral proteins known as pathogenicity determinants have evolved to coordinate a network of protein interactions. The βC1 protein encoded by specific geminiviral satellites acts as a key pathogenicity determinant for this disease-causing family of plant viruses. Post-translational modifications (PTMs) such as ubiquitination and phosphorylation of the βC1 protein have been shown to occur in diverse viruses. However, the relevance of these and other layers of PTMs in host-geminiviral interactions has not been fully understood.

RESULTS: Here we identified the significance of a novel layer of PTMs in the βC1 protein of Synedrella yellow vein clearing virus (SyYVCV), a newly identified member of the Begomovirus genus of Geminiviruses. This protein has conserved SUMOylation and SUMO-interacting motifs (SIMs), and we observed SUMOylation of SyYVCV βC1 in host plants as a defensive strategy against ubiquitin-mediated degradation. Counteracting this, SIMs encoded in βC1 mediate the degradation of βC1; however, both these PTMs are essential for the function of βC1 protein since SIM and SUMOylation motif mutants failed to promote pathogenicity and viral replication in vivo. SUMOylation in different motifs of βC1 led to functionally distinct outcomes, regulating the stability and function of the βC1 protein, as well as increased global SUMOylation of host proteins.

CONCLUSION: Our results indicate the presence of a novel mechanism mediating a fine balance between defence and counter-defence in which a SIM site is competitively sought for degradation and, as a counter-defence, βC1 undergoes SUMOylation to escape from its degradation.

}, issn = {1741-7007}, doi = {10.1186/s12915-020-00843-y}, author = {Nair, Ashwin and Chatterjee, Kiran Sankar and Jha, Vikram and Das, Ranabir and Shivaprasad, P V} } @article {127107, title = {Extraction and Purification of Laccases from Rice Stems.}, journal = {Bio Protoc}, volume = {9}, year = {2019}, month = {2019 Apr 05}, pages = {e3208}, abstract = {

Laccases are found in cell walls of plants in very low amounts. This protocol provides an efficient method to purify laccases from rice stems. The method involves three steps: 1) Isolation of total protein from rice stems using buffers with high salt concentration to extract protein from cell walls; 2) Purification of laccases using concanavalin-A beads; and, 3) In-gel staining of laccases with 4-hydroxyindole. Concanavalin-A specifically binds to internal or non-reducing terminal α-D-mannosyl and α-D-glucosyl groups found in glycoproteins and glycolipids. Laccases being glycoproteins binds to concanavalin-A during purification process and eluted with mannose.

}, issn = {2331-8325}, doi = {10.21769/BioProtoc.3208}, author = {Swetha, Chenna and Shivaprasad, P V} } @article {59710, title = {miR828 and miR858 regulate VvMYB114 to promote anthocyanin and flavonol accumulation in grapes.}, journal = {J Exp Bot}, year = {2019}, month = {2019 May 30}, abstract = {

MicroRNAs are a class of non-coding small RNAs involved in negative regulation of gene expression and playing critical roles in developmental and metabolic pathways. Studies in several plants have identified few microRNAs and other small (s) RNAs that target regulators of phenylpropanoid metabolic pathway called MYB transcription factors. However, it is not well-understood how sRNA mediated regulation of MYBs influences accumulation of specific secondary metabolites. Using sRNA-sequencing, degradome analysis, mRNA-seq and proteomic analysis, we establish that grape lines with high anthocyanin content express two abundant MYB-targeting miRNAs resulting in differential expression of specific MYB proteins. miR828 and miR858 target coding sequences of specific helix motifs in the mRNA sequences of MYB proteins. Targeting by miR828, caused MYB RNA decay and production of cascade secondary siRNAs that depend on RNA dependant RNA polymerase 6. MYB suppression and cascade silencing was more robust in grape lines with high anthocyanin content when compared to a flavonol-rich grape line. We establish that miRNA-mediated silencing targeted repressor class of MYBs to promote anthocyanin biosynthesis in grape lines with high anthocyanins. We propose that this process regulates expression of appropriate MYBs in grape lines to produce specific secondary metabolites.

}, issn = {1460-2431}, doi = {10.1093/jxb/erz264}, author = {Tirumalai, Varsha and Swetha, Chenna and Nair, Ashwin and Pandit, Awadhesh and Shivaprasad, P V} } @article {34252, title = {Molecular characterization of a new begomovirus infecting Synedrella nodiflora in South India.}, journal = {Arch Virol}, year = {2018}, month = {2018 May 09}, abstract = {

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.

}, issn = {1432-8798}, doi = {10.1007/s00705-018-3861-3}, author = {Das, Soumita and Hegde, Akshay and Shivaprasad, P V} } @inbook {16850, title = {Artificial Induction and Maintenance of Epigenetic Variations in Plants}, booktitle = {Plant Gene Silencing: Mechanisms and Applications}, year = {2017}, pages = {59-75}, publisher = {CAB International}, organization = {CAB International}, chapter = {3}, isbn = {9781780647678}, author = {Das, Soumita and Singh, Rahul Raj and Shivaprasad, P V} } @article {16579, title = {Methylglyoxal detoxification by a DJ-1 family protein provides dual abiotic and biotic stress tolerance in transgenic plants.}, journal = {Plant Mol Biol}, year = {2017}, month = {2017 Apr 25}, abstract = {

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.

}, issn = {1573-5028}, doi = {10.1007/s11103-017-0613-9}, author = {Melvin, Prasad and Bankapalli, Kondalarao and D{\textquoteright}Silva, Patrick and Shivaprasad, P V} }