Metagenomics and metatranscriptomics as potential driving forces for the exploration of diversity and functions of micro-eukaryotes in soil.

Micro-eukaryotes are ubiquitous and play vital roles in diverse ecological systems, yet their diversity and functions are scarcely known. This may be due to the limitations of formerly used conventional culture-based methods. Metagenomics and metatranscriptomics are enabling to unravel the genomic, metabolic, and phylogenetic diversity of micro-eukaryotes inhabiting in different ecosystems in a more comprehensive manner. The in-depth study of structural and functional characteristics of micro-eukaryote community residing in soil is crucial for the complete understanding of this major ecosystem. This review provides a deep insight into the methodologies employed under these approaches to study soil micro-eukaryotic organisms. Furthermore, the review describes available computational tools, pipelines, and database sources and their manipulation for the analysis of sequence data of micro-eukaryotic origin. The challenges and limitations of these approaches are also discussed in detail. In addition, this review summarizes the key findings of metagenomic and metatranscriptomic studies on soil micro-eukaryotes. It also highlights the exploitation of these methods to study the structural as well as functional profiles of soil micro-eukaryotic community and to screen functional eukaryotic protein coding genes for biotechnological applications along with the future perspectives in the field.

Metagenomics analysis reveals a new metallothionein family: Sequence and metal-binding features of new environmental cysteine-rich proteins.

Metallothioneins are cysteine-rich proteins, which function as (i) metal carriers in basal cell metabolism and (ii) protective metal chelators in conditions of metal excess. Metallothioneins have been characterized from different eukaryotic model and cultivable species. Presently, they are categorized in 15 families but evolutionary relationships between these metallothionein families remain unresolved. Several cysteine-rich protein encoding genes that conferred Cd-tolerance in Cd-sensitive yeast mutants have previously been isolated from soil eukaryotic metatranscriptomes. They were called CRPs for "cysteine-rich proteins". These proteins, of unknown taxonomic origins, share conserved cysteine motifs and could be considered as metallothioneins. In the present work, we analyzed these CRPs with respect to their amino acid sequence features and their metal-binding abilities towards Cd, Zn and Cu metal ions. Sequence analysis revealed that they share common features with different known metallothionein families, but also exhibit unique specific features. Noticeably, CRPs display two separate cysteine-rich domains which, when expressed separately in yeast, confer Cd-tolerance. The N-terminal domain contains some conserved atypical Cys motifs, such as one CCC and two CXCC ones. Five CRPs were expressed and purified as recombinant proteins and their metal-binding characteristics were studied. All these CRPs chelated Cd(II), Zn(II) and Cu(I), although displaying a better capacity for Zn(II) coordination. All CRPs are able to confer Cd-tolerance, and four of them confer Zn-tolerance in the Zn-sensitive zrc1Δ yeast mutant. We designated these CRPs as environmental metallothioneins belonging to a new formerly undescribed metallothionein family.

Construction of sized eukaryotic cDNA libraries using low input of total environmental metatranscriptomic RNA.

BACKGROUND: Construction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest. Many of the inserts in libraries constructed by standard methods are represented by truncated cDNAs due to premature stoppage of reverse transcriptase activity and preferential cloning of short cDNAs. RESULTS: We report here a simple and cost effective technique for preparation of sized eukaryotic cDNA libraries from as low as three microgram of total soil RNA dominated by ribosomal and bacterial RNA. cDNAs synthesized by a template switching approach were size-fractionated by two dimensional agarose gel electrophoresis prior to PCR amplification and cloning. Effective size selection was demonstrated by PCR amplification of conserved gene families specific of each size class. Libraries of more than one million independent inserts whose sizes ranged between one and four kb were thus produced. Up to 80% of the insert sequences were homologous to eukaryotic gene sequences present in public databases. CONCLUSIONS: A simple and cost effective technique has been developed to construct sized eukaryotic cDNA libraries from environmental samples. This technique will facilitate expression cloning of environmental eukaryotic genes and contribute to a better understanding of basic biological and/or ecological processes carried out by eukaryotic microbial communities.

Differential soybean gene expression during early phase of infection with Mungbean yellow mosaic India virus.

Mungbean yellow mosaic India virus (MYMIV), a bipartite begomovirus, causes yellow mosaic disease to soybean. Studies related to host gene expression in response to begomovirus infection have mostly been performed with systemically infected tissues at a later period of infection. In this study, soybean gene expression analysis has been performed to understand local responses against MYMIV at an early stage of infection before appearance of detectable limit of late viral transcripts. 444 soybean transcripts belonging to eleven functional categories showed significant changes in expression level at two days after infection. MYMIV infection resulted in enhanced expression of genes associated with hypersensitive response, programmed cell death and resistance response pathways and reduced expression of genes for photosynthesis and sugar transport. Comparative expression analysis of selected transcripts in the susceptible and a resistant variety displayed differential expression of host genes involved in intercellular virus movement and long distance signaling of systemic acquired resistance.

Enhanced viral intergenic region-specific short interfering RNA accumulation and DNA methylation correlates with resistance against a geminivirus.

RNA silencing mediated by short-interfering RNA (siRNA) is used by plants as a defense against viruses. In the case of geminiviruses, viral DNA is targeted at the transcriptional level, while virus-derived transcripts are targeted by posttranscriptional silencing. Mungbean yellow mosaic India virus (MYMIV), a bipartite geminivirus, causes yellow mosaic disease in soybean (Glycine max). A soybean variety resistant to this disease has been identified (line PK416). To understand the molecular mechanism underlying this resistance, distribution of MYMIV-derived siRNAs along the viral genome was compared in resistant and susceptible plants, using samples obtained in the first few days following inoculation. We observed that, in the resistant soybean variety, most of the virus-derived siRNAs were complementary to the intergenic region (IR), while in the susceptible variety (line JS335), a majority of the siRNAs corresponded to coding regions of the viral genome. Most of the IR-specific siRNA molecules produced in the resistant plants were 24 nt in size. Bisulfite sequencing showed that, in the resistant plants, a higher level of methylation occurred in the IR of viral DNA.

CAP2 enhances germination of transgenic tobacco seeds at high temperature and promotes heat stress tolerance in yeast.

We reported earlier that ectopic expression of CAP2, a single AP2 domain containing transcription activator from chickpea (Cicer arietinum) in tobacco improves growth and development, and tolerance to dehydration and salt stress, of the transgenic plants. Here, we report that, in addition, the CAP2-transgenic tobacco seeds also exhibit higher germination efficiency at high temperature and show higher expression levels of genes for tobacco heat shock proteins and a heat shock factor. CAP2 was able to activate the 5'-upstream activating sequence of tobacco heat shock factor. Surprisingly, expression of CAP2 cDNA in Saccharomyces cerevisiae also enhanced heat tolerance, with increased expression of the gene for yeast heat shock factor 1 (Hsf1) and its target, the gene for yeast heat shock protein 104 (Hsp104). Sequence analysis of the Hsf1 promoter revealed the presence of a dehydration-responsive element/C-repeat-like element (DRE/CRE). Recombinant CAP2 protein bound to the DRE/CRE in the Hsf1 promoter in a gel shift assay and transactivated the Hsf1 promoter-His reporter construct. The full-length CAP2 protein was required to provide thermotolerance in yeast. If these findings are taken together, our results suggest that CAP2 is involved in the heat stress response and provides an example of functioning of a plant transcription factor in yeast, highlighting the strong evolutionary conservation of the stress response mechanism.

Soybean cultivar resistant to Mungbean Yellow Mosaic India Virus infection induces viral RNA degradation earlier than the susceptible cultivar.

Yellow mosaic disease caused by whitefly-transmitted bipartite Geminiviruses is one of the major constraints on productivity of a number of pulse crops. We have cloned the bipartite genome of Mungbean Yellow Mosaic India Virus isolated from infected Soybean. We report here that agroinfection of Soybean seedlings with a single uncut recombinant binary plasmid containing tandem dimers of both DNA A and DNA B resulted in 100% infectivity in susceptible varieties. To understand the mechanism of natural resistance in a Soybean variety, we compared the abundance of the viral RNAs in a resistant and a susceptible variety at the early time points after agroinfection. Whilst the resistant variety displayed synthesis but rapid degradation of the early viral RNAs; the degradation in the susceptible variety was delayed resulting in accumulation of those transcripts later in infection. Accumulation of the late viral transcripts and DNA replication were detectable only in the susceptible variety. This indicates that rapid degradation of the early viral transcripts, possibly through siRNA mechanism, is one of the probable mechanisms of natural resistance against geminivirus.