@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 {162685, title = {Loss of function of chromatin remodeler OsCLSY4 leads to RdDM-mediated mis-expression of endosperm-specific genes affecting grain qualities.}, journal = {PLoS Genet}, volume = {21}, year = {2025}, month = {2025 Dec}, pages = {e1011956}, abstract = {

In plants, RNA-directed DNA methylation (RdDM) sequence-specifically targets transposable elements (TEs) and repeats, often in a tissue-specific manner. In triploid endosperm tissue, RdDM also acts as a parental dosage regulator, mediating spatio-temporal expression of genes required for its development. It is unclear how RdDM is initiated and established in endosperm. Rice endosperm-specific imprinted chromatin remodeler OsCLSY3 recruits RNA polymerase IV to specific genomic sites for silencing and optimal gene expression. Here we show that, in addition to OsCLSY3, ubiquitously expressed OsCLSY4 is also crucial for proper reproductive growth and endosperm development. Loss of function of OsCLSY4 led to reproductive and nutrient-filling defects in endosperm. Using genetic and molecular analysis, we show that both OsCLSY3 and OsCLSY4 play overlapping and unique silencing roles in rice endosperm, by targeting specific and shared genomic regions such as TEs, repeats and genic regions. These results indicate the importance of optimal expression of two OsCLSYs in regulating endosperm-specific gene expression, genomic imprinting and suppression of specific TEs. Results presented here provide new insights into the functions of rice CLSYs as upstream RdDM regulators in rice endosperm development, and we propose that functions of their homologs might be conserved across monocots.

}, keywords = {Chromatin Assembly and Disassembly, DNA Methylation, DNA Transposable Elements, Edible Grain, Endosperm, Gene Expression Regulation, Plant, Genomic Imprinting, Oryza, Plant Proteins}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1011956}, author = {Pal, Avik Kumar and Rana, Saumyapriya and Dey, Riju and Shivaprasad, Padubidri V} } @article {160480, title = {OsWRKY53 dictates wound responses in rice through fine-tuning crosstalk between PEP and PSK-mediated signalling.}, journal = {Plant J}, volume = {123}, year = {2025}, month = {2025 Sep}, pages = {e70481}, abstract = {

Wounding is a major event during the multitude of stresses that plants face in their natural environment. Wound response is very dynamic and involves the integration of various regulatory networks culminating in successful wound-induced downstream signalling. Plants depend on endogenous molecular signals predominantly, small peptides, to initiate wound responses. Transcriptional response is paramount in dictating a successful wound response. Here we show the involvement of a WRKY transcription factor (TF) named OsWRKY53 that is upregulated upon wounding as well as wound-derived plant elicitor peptide (PEP), OsPep2 treatments. OsWRKY53 is involved in the positive regulation of gene expression of OsPep2-responsive genes. OsWRKY53 displays altered DNA occupancy in response to OsPep2 treatment over time, correlating with the altered gene expression. OsWRKY53 undergoes phosphorylation upon OsPep2 perception, and this contributes to the enhanced stability of the protein. Further, OsWRKY53 is involved in the simultaneous activation and suppression of OsPep2-responsive and phytosulfokine (PSK)-responsive genes, respectively. In agreement with these findings, perturbed expression of OsWRKY53 leads to compromised transcriptional responses post-wounding. Collectively, we establish that OsWRKY53 acts at the intersection of PEP and PSK-mediated transition of wound responses.

}, keywords = {Gene Expression Regulation, Plant, Oryza, Phosphorylation, Plant Proteins, Signal Transduction, Transcription Factors}, issn = {1365-313X}, doi = {10.1111/tpj.70481}, author = {Harshith, Chitthavalli Y and Palchaudhuri, Dipasmit and Dey, Riju and Raju, Steffi and Shivaprasad, Padubidri V} } @article {149951, title = {Genome-wide association study reveals effect of nsSNPs on candidate genes in rice during iron deficiency.}, journal = {Funct Integr Genomics}, volume = {24}, year = {2024}, month = {2024 Oct 25}, pages = {198}, abstract = {

Resource-poor areas with moisture deficit lands following aerobic and direct seeded rice (DSR) methods of cultivation face severe problems of iron deficiency. In this study, Bengal and Assam Aus rice panel was phenotyped at the seedling stage using an iron-deprived hydroponic medium for various shoot and root traits. A novel iron deficiency scoring scale was used to classify the tolerance reaction and could range anywhere between 0 and 9, indicating the most tolerant and susceptible, respectively. The GWAS results identified four putative candidate genes; OsFLA for number of leaves and shoot length, OsBIDK1 for root traits; average diameter, volume, biomass, projected area, and surface area, OsHPL3 for chlorophyll index of the third leaf and AKR2B (XBOS252) was for Fe score, (which was earlier reported in relation to Xa21). The nsSNP (nsSNPs) variations in these gene sequences were used to group the panel and identify superior haplotypes and donors. BR16 was identified as a superior donor, with higher chlorophyll index and shoot length than RA23, also higher values for root traits like root average diameter, root volume, root projected area and root surface area followed by Shete Bhado. The impact of identified nsSNPs on protein structure and stability was investigated. The conserved domains detected in the mutated proteins of the superior haplotypes are very informative, highlighting that natural selection favors abiotic stress tolerant variants in resource poor areas. Thus, justifying our choice of Aus landraces for association mapping of Fe deficiency tolerant genes in rice.

}, keywords = {Chlorophyll, Genome-Wide Association Study, Iron Deficiencies, Oryza, Plant Proteins, Plant Roots, Polymorphism, Single Nucleotide}, issn = {1438-7948}, doi = {10.1007/s10142-024-01478-w}, author = {Panda, Siddharth and Anandan, Annamalai and Shafi, K Mohamed and Naika, Mahantesha B N and Sowdhamini, R and Vanishree, G and Sarkar, Sutapa and Travis, Anthony J and Norton, Gareth J and Price, Adam H} } @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 {147285, title = {Wound-induced small-peptide-mediated signaling cascade, regulated by OsPSKR, dictates balance between growth and defense in rice.}, journal = {Cell Rep}, volume = {43}, year = {2024}, month = {2024 Jul 23}, pages = {114515}, abstract = {

Wounding is a general stress in plants that results from various pest and pathogenic infections in addition to environment-induced mechanical damages. Plants have sophisticated molecular mechanisms to recognize and respond to wounding, with those of monocots being distinct from dicots. Here, we show the involvement of two distinct categories of temporally separated, endogenously derived peptides, namely, plant elicitor peptides (PEPs) and phytosulfokine (PSK), mediating wound responses in rice. These peptides trigger a dynamic signal relay in which a receptor kinase involved in PSK perception named OsPSKR plays a major role. Perturbation of OsPSKR expression in rice leads to compromised development and constitutive autoimmune phenotypes. OsPSKR regulates the transitioning of defense to growth signals upon wounding. OsPSKR displays mutual antagonism with the OsPEPR1 receptor involved in PEP perception. Collectively, our work indicates the presence of a stepwise peptide-mediated signal relay that regulates the transition from defense to growth upon wounding in monocots.

}, keywords = {Gene Expression Regulation, Plant, Oryza, Peptides, Plant Diseases, Plant Proteins, Signal Transduction}, issn = {2211-1247}, doi = {10.1016/j.celrep.2024.114515}, author = {Harshith, Chitthavalli Y and Pal, Avik and Chakraborty, Monoswi and Nair, Ashwin and Raju, Steffi and Shivaprasad, Padubidri V} } @article {124206, title = {A conserved SNP variation in the pre-miR396c flanking region in Oryza sativa indica landraces correlates with mature miRNA abundance.}, journal = {Sci Rep}, volume = {13}, year = {2023}, month = {2023 Feb 07}, pages = {2195}, abstract = {

Plant precursor miRNAs (pre-miRNA) have conserved evolutionary footprints that correlate with mode of miRNA biogenesis. In plants, base to loop and loop to base modes of biogenesis have been reported. Conserved structural element(s) in pre-miRNA play a major role in turn over and abundance of mature miRNA. Pre-miR396c sequences and secondary structural characteristics across Oryza species are presented. Based on secondary structure, twelve Oryza pre-miR396c sequences are divided into three groups, with the precursor from halophytic Oryza coarctata forming a distinct group.\ The miRNA-miRNA* duplex region is completely conserved across eleven\ Oryza\ species as are other structural elements in the pre-miRNA, suggestive of an evolutionarily conserved base-to-loop mode of miRNA biogenesis. SNPs within O. coarctata mature miR396c sequence and miRNA* region have the potential to alter target specificity and association with the RNA-induced silencing complex. A conserved SNP variation, rs10234287911 (G/A), identified in O. sativa pre-miR396c sequences alters base pairing above the miRNA-miRNA* duplex. The more stable structure conferred by the {\textquoteright}A{\textquoteright} allele may promote better processing vis-{\`a}-vis the structure conferred by {\textquoteright}G{\textquoteright} allele. We also examine pri- and pre-miR396c expression in cultivated rice under heat and salinity and their correlation with miR396c expression.

}, keywords = {MicroRNAs, Oryza, Polymorphism, Single Nucleotide, Salt-Tolerant Plants}, issn = {2045-2322}, doi = {10.1038/s41598-023-28836-1}, author = {Jaganathan, Deepa and Rajakani, Raja and Doddamani, Dadakhalandar and Saravanan, Divya and Pulipati, Shalini and Vivek Hari Sundar G and Sellamuthu, Gothandapani and Jayabalan, Shilpha and Kumari, Kumkum and Parthasarathy, Pavithra and S, Punitha and Ramalingam, Sivaprakash and Shivaprasad, Padubidri V and Venkataraman, Gayatri} } @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} }