MET18 Connects the Cytosolic Iron-Sulfur Cluster Assembly Pathway to Active DNA Demethylation in Arabidopsis.

DNA demethylation mediated by the DNA glycosylase ROS1 helps determine genomic DNA methylation patterns and protects active genes from being silenced. However, little is known about the mechanism of regulation of ROS1 enzymatic activity. Using a forward genetic screen, we identified an anti-silencing (ASI) factor, ASI3, the dysfunction of which causes transgene promoter hyper-methylation and silencing. Map-based cloning identified ASI3 as MET18, a component of the cytosolic iron-sulfur cluster assembly (CIA) pathway. Mutation in MET18 leads to hyper-methylation at thousands of genomic loci, the majority of which overlap with hypermethylated loci identified in ros1 and ros1dml2dml3 mutants. Affinity purification followed by mass spectrometry indicated that ROS1 physically associates with MET18 and other CIA components. Yeast two-hybrid and split luciferase assays showed that ROS1 can directly interact with MET18 and another CIA component, AE7. Site-directed mutagenesis of ROS1 indicated that the conserved iron-sulfur motif is indispensable for ROS1 enzymatic activity. Our results suggest that ROS1-mediated active DNA demethylation requires MET18-dependent transfer of the iron-sulfur cluster, highlighting an important role of the CIA pathway in epigenetic regulation.

The complete genome sequence of a south Indian isolate of Rice tungro spherical virus reveals evidence of genetic recombination between distinct isolates.

In this study, complete genome of a south Indian isolate of Rice tungro spherical virus (RTSV) from Andhra Pradesh (AP) was sequenced, and the predicted amino acid sequence was analysed. The RTSV RNA genome consists of 12,171 nt without the poly(A) tail, encoding a putative typical polyprotein of 3,470 amino acids. Furthermore, cleavage sites and sequence motifs of the polyprotein were predicted. Multiple alignment with other RTSV isolates showed a nucleotide sequence identity of 95% to east Indian isolates and 90% to Philippines isolates. A phylogenetic tree based on complete genome sequence showed that Indian isolates clustered together, while Vt6 and PhilA isolates of Philippines formed two separate clusters. Twelve recombination events were detected in RNA genome of RTSV using the Recombination Detection Program version 3. Recombination analysis suggested significant role of 5' end and central region of genome in virus evolution. Further, AP and Odisha isolates appeared as important RTSV isolates involved in diversification of this virus in India through recombination phenomenon. The new addition of complete genome of first south Indian isolate provided an opportunity to establish the molecular evolution of RTSV through recombination analysis and phylogenetic relationship.

Genomics of helper component proteinase reveals effective strategy for papaya ringspot virus resistance.

Papaya ringspot virus (PRSV) causes severe economic losses in both cucurbits and papaya throughout the tropics and subtropics. Development of PRSV-resistant transgenic plants faces a major hurdle in achieving resistance against geographically distinct isolates. One of the major reasons of failing to achieve the broad-spectrum PRSV resistance is the involvement of silencing suppressor proteins of viral origin. Here, based on sequence profile of silencing suppressor protein, HcPro, we show that PRSV-HcPro, acts as a suppressor of RNA silencing through micro RNA binding in a dose- dependent manner. In planta expression of PRSV-HcPro affects developmental biology of plants, suggesting the interference of suppressor protein in micro RNA-directed regulatory pathways of plants. Besides facilitating the establishment of PRSV, it showed strong positive synergism with other heterologous viruses as well. This study provides a strategy to develop effective and stable PRSV-resistant transgenic plants.

Ambient temperature perception in papaya for papaya ringspot virus interaction.

Temperature dramatically affects the host-virus interaction. Outbreaks of viral diseases are frequently associated with the ambient temperature required for host development. Using papaya as a host and Papaya ringspot virus (PRSV) as a pathogen, we studied the effect of temperature on the intensity of disease symptoms and virus accumulation. The phenotypic expression of symptoms and viral accumulation were found to be maximum at ambient temperature (26-31 degrees C) of papaya cultivation. However, there was a drastic difference, 10 degrees C above and below the ambient temperature. The underlying mechanism of these well-known observations are not yet understood completely; however, these observations might help find answers in RNA silencing mechanism of plants. Since viral-derived silencing suppressor proteins play a significant role in RNA silencing mechanism, here we show that PRSV-derived Helper component proteinase (HC-Pro) protein has an affinity for small RNAs in a temperature-dependent manner. This suggested the probable role of HC-Pro in the temperature-regulated host-virus relationship.

Role of genetic recombination in the molecular architecture of Papaya ringspot virus.

Papaya ringspot virus (PRSV) has a single-stranded RNA genome and causes severe economic losses both in cucurbits and papaya worldwide. The extent to which the genome of PRSV is shaped by recombination provides an understanding of the molecular evolution of PRSV and helps in studying features such as host specificity, geographic distribution, and its emergence as new epidemics. The PRSV-P-Indian isolate was completely sequenced and compared with 14 other isolates reported from the rest of the world for their phylogenetic survey of recombination events. Cistron-by-cistron sequence comparison and phylogenetic analysis based on full-genome polyprotein showed two distinct groupings of Asian and American isolates, although PRSV-P and W-India clustered along with the American isolates. Recombination sites were found throughout the genomes, except in the small 6K1 protein gene. A significant proportion of recombination hotspots was found in the P1 gene, followed by P3, cylindrical inclusion (CI), and helper component proteinase (HcPro). Correlations between the presence of recombination sites, geographic distribution, and phylogenetic relationship provide an opportunity to establish the molecular evolution and geographic route of PRSV.

Behavior of RNAi suppressor protein 2b of Cucumber mosaic virus in planta in presence and absence of virus.

The 2b protein encoded by Cucumber mosaic virus (CMV) has been shown as a virus counter defense factor that interferes with the RNAi pathway. The 2b gene from CMV-banana, New Delhi isolate (CMV-NDLS) was amplified from CMV infected cucumber plants to generate the sense and antisense binary vector constructs for 2b expression and repression in planta. Constitutive expression of 2b gene in healthy Nicotiana tabacum caused phenotypic aberrations during somatic embryogenesis, which were not observed when expressed in CMV infected N. tabacum. Further, the established virus population in CMV infected N. tabacum was not affected by constitutive expression and repression of 2b gene. Thus, indicating its role in initiation of gene silencing, at the early stage of viral infection. This is the first demonstration of differential behavior of 2b suppressor protein in host development in the absence and presence of virus.