T. Vinutha

Modulation of Plant Development and MYB Down Regulation: Both during In Planta Expression of miR159a and in Natural ToLCV Infection

Viral infections result in developmental abnormalities. Symptomatic expression of Tomato leaf curl virus (ToLCV) in tomato, results in curling of leaves and withering of flowers. These modulations are due to altered host gene expression. Transcription factors (TF) are key elements, which control gene expression. In this study, we observed accumulation of miRNA159a levels, responsible for regulating MYB-TF during ToLCV infection. The MYB transcription factor control gene expression of various components involved in floral, anther and leaf development. During ToLCV infection two-fold accumulation of miR159a was observed leading to lower levels of MYB. Transgenic Nicotiana tabacum expressing pre-miR159a mimics ToLCV infection. This suggests probable role of miR159a driven gene regulatory pathway responsible for appearance of viral symptoms.

Enhanced nutraceutical potential of gamma irradiated black soybean extracts.

Radiation processing of soybean, varying in seed coat colour, was carried out at dose levels of 0.25, 0.5 and 1 kGy to evaluate their potential anti-proliferative and cytoprotective effects in an in vitro cell culture system. Irradiated and control black (Kalitur) and yellow (DS9712) soybean extracts were characterized in terms of total phenolics, flavonoids and anthocyanins, especially cyanidin-3-glucoside (C3G). Using an epithelial cell line, BEAS-2B the potential cytoprotective effects of soybean extracts were evaluated in terms of intracellular ROS levels and cell viability. The most relevant scavenging effect was found in Kalitur, with 78% decrease in ROS, which well correlated with a 33% increase in C3G after a 1 kGy dose. Results evidenced a correspondence between in vitro antioxidant activity and a potential health property of black soybean extracts, exemplifying the nutraceutical role of C3G. To our knowledge this study is the first report validating the cytoprotective effects of irradiated black soybean extracts.

Exploring the role of Inositol 1,3,4-trisphosphate 5/6 kinase-2 (GmITPK2) as a dehydration and salinity stress regulator in Glycine max (L.) Merr. through heterologous expression in E. coli

Phytic acid (PA) is implicative in a spectrum of biochemical and physiological processes involved in plant stress response. Inositol 1,3,4, Tris phosphate 5/6 kinase (ITPK), a polyphosphate kinase that converts Inositol 1,3,4 trisphosphate to Inositol 1,3,4,5/6 tetra phosphate, averting the inositol phosphate pool towards PA biosynthesis, is a key regulator that exists in four different isoforms in soybean. In the present study, in-silico analysis of the promoter region of ITPKs was done and among the four isoforms, promoter region of GmITPK2 showed the presence of two MYB binding elements for drought inducibility and one for ABA response. Expression profiling through qRT-PCR under drought and salinity stress showed higher expression of GmITPK2 isoform compared to the other members of the family. The study revealed GmITPK2 as an early dehydration responsive gene which is also induced by dehydration and exogenous treatment with ABA. To evaluate the osmo-protective role of GmITPK2, attempts were made to assess the bacterial growth on Luria Broth media containing 200 mM NaCl, 16% PEG and 100 μM ABA, individually. The transformed E. coli BL21 (DE3) cells harbouring the GmITPK2 gene depicted better growth on the media compared to the bacterial cells containing the vector alone. Similarly, the growth of the transformed cells in the liquid media containing 200 mM NaCl, 16% PEG and 100 μM ABA showed higher absorbance at 600 nm compared to control, at different time intervals. The GmITPK2 recombinant E. coli cells showing tolerance to drought and salinity thus demonstrated the functional redundancy of the gene across taxa. The purity and specificity of the recombinant protein was assessed and confirmed through PAGE showing a band of ∼35 kDa on western blotting using Anti- Penta His- HRP conjugate antibody. To the best of our knowledge, the present study is the first report exemplifying the role of GmITPK2 isoform in drought and salinity tolerance in soybean.

Tomato geminivirus encoded RNAi suppressor protein, AC4 interacts with host AGO4 and precludes viral DNA methylation

Plant RNA silencing systems are organized as a network, regulating plant developmental pathways and restraining invading viruses, by sharing cellular components with overlapping functions. Host regulatory networks operate either at the transcriptional level via RNA-directed DNA methylation, or at the post-transcriptional stage interfering with mRNA to restrict viral infection. However, viral-derived proteins, including suppressors of RNA silencing, favour virus establishment, and also affect plant developmental processes. In this investigation, we report that Tomato leaf curl New Delhi virus-derived AC4 protein suppresses RNA silencing activity and mutational analysis of AC4 showed that Asn-50 in the SKNT-51 motif, in the C-terminal region, is a critical determinant of its RNA silencing suppressor activity. AC4 showed interaction with host AGO4 but not with AGO1, aggregated around the nucleus, and influenced cytosine methylation of the viral genome. The possible molecular mechanism by which AC4 interferes in the RNA silencing network, helps virus establishment, and affects plant development is discussed.

Comparative analysis of tocopherol biosynthesis genes and its transcriptional regulation in soybean seeds.

Tocopherols composed of four isoforms (α, β, γ, and δ) and its biosynthesis comprises of three pathways: methylerythritol 4-phosphate (MEP), shikimate (SK) and tocopherol-core pathways regulated by 25 enzymes. To understand pathway regulatory mechanism at transcriptional level, gene expression profile of tocopherol-biosynthesis genes in two soybean genotypes was carried out, the results showed significantly differential expression of 5 genes: 1-deoxy-d-xylulose-5-P-reductoisomerase (DXR), geranyl geranyl reductase (GGDR) from MEP, arogenate dehydrogenase (TyrA), tyrosine aminotransferase (TAT) from SK and γ-tocopherol methyl transferase 3 (γ-TMT3) from tocopherol-core pathways. Expression data were further analyzed for total tocopherol (T-toc) and α-tocopherol (α-toc) content by coregulation network and gene clustering approaches, the results showed least and strong association of γ-TMT3/tocopherol cyclase (TC) and DXR/DXS, respectively, with gene clusters of tocopherol biosynthesis suggested the specific role of γ-TMT3/TC in determining tocopherol accumulation and intricacy of DXR/DXS genes in coordinating precursor pathways toward tocopherol biosynthesis in soybean seeds. Thus, the present study provides insight into the major role of these genes regulating the tocopherol synthesis in soybean seeds.

Possible role of miRNAs and their targets, in modulating leaf morphology and plant growth during leaf curl virus infection in tomato

Small RNAs (sRNAs) play important roles in all stages of plant growth and development. Several classes of sRNAs have been described; the best-described classes are referred to as microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs are small endogenous RNAs that regulate gene expression by mainly targeting the transcription factors in plants and animals. miRNAs are involved in a variety of activities, including plant development, signal transduction, protein degradation, response to environmental stress, and pathogen invasion. Virus infection is one of the biotic stresses, which plant face very frequently. Tomato leaf curl New Delhi viruses (ToLCNDVs) are single standard DNA viruses belong to the family Geminirividae. It infects tomato plants worldwide causing leaf curling, and is responsible for heavy economic losses to tomato production. Two strains of ToLCNDV exist in nature, one showing severe leaf curling symptom and another showing mild symptom after infection. To understand this difference, we analyzed miRNA expression levels using microarray. Expression levels of fourteen miRNAs were found to be differentially regulated in the plants inoculated with severe and mild strain of ToLCNDV. Among them, eleven miRNAs are involved in organ development, hormone signaling and defense responses through their targets. Four constitutive differentially regulated miRNAs (miR-160, 164, 167 and 390) through their targets (viz. ARF8, NAC, ARF3 and MAPK, respectively) control leaf development. Stress responsive miR-403, through its target AGO2, regulates RNAi-mediated defense response. In the present study, we discuss the possible interference of viral proteins in plant development process through the miRNAs.

Molecular mechanism of Begomovirus evolution and plant defense response

Abstract Viruses depend exclusively on the host cell machinery for their propagation and survival, hence they modulate the host gene expression to suit their needs. The range of interactions between various host and viral factors leads to developmental abnormalities in the host. These interactions can be fine-tuned in the host by modulating gene expression through triggering a defense mechanism and allowing epigenetic modifications. In viral genomes a high rate of mutation adds to the complexity of these interactions and helps in developing adaptation to different hosts.