Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence.

Kinetochores facilitate interaction between chromosomes and the spindle apparatus. The formation of a metazoan trilayered kinetochore is an ordered event in which inner, middle, and outer layers assemble during disassembly of the nuclear envelope during mitosis. The existence of a similar strong correlation between kinetochore assembly and nuclear envelope breakdown in unicellular eukaryotes is unclear. Studies in the hemiascomycetous budding yeasts Saccharomyces cerevisiae and Candida albicans suggest that an ordered kinetochore assembly may not be evolutionarily conserved. Here, we utilized high-resolution time-lapse microscopy to analyze the localization patterns of a series of putative kinetochore proteins in the basidiomycetous budding yeast Cryptococcus neoformans, a human pathogen. Strikingly, similar to most metazoa but atypical of yeasts, the centromeres are not clustered but positioned adjacent to the nuclear envelope in premitotic C. neoformans cells. The centromeres gradually coalesce to a single cluster as cells progress toward mitosis. The mitotic clustering of centromeres seems to be dependent on the integrity of the mitotic spindle. To study the dynamics of the nuclear envelope, we followed the localization of two marker proteins, Ndc1 and Nup107. Fluorescence microscopy of the nuclear envelope and components of the kinetochore, along with ultrastructure analysis by transmission electron microscopy, reveal that in C. neoformans, the kinetochore assembles in an ordered manner prior to mitosis in concert with a partial opening of the nuclear envelope. Taken together, the results of this study demonstrate that kinetochore dynamics in C. neoformans is reminiscent of that of metazoans and shed new light on the evolution of mitosis in eukaryotes.

The centromere, on which kinetochore proteins assemble, ensures precise chromosome segregation. Centromeres are largely specified by the histone H3 variant CENP-A (also known as Cse4 in yeasts). Structurally, centromere DNA sequences are highly diverse in nature. However, the evolutionary consequence of these structural diversities on de novo CENP-A chromatin formation remains elusive. Here, we report the identification of centromeres, as the binding sites of four evolutionarily conserved kinetochore proteins, in the human pathogenic budding yeast Candida tropicalis. Each of the seven centromeres comprises a 2 to 5 kb non-repetitive mid core flanked by 2 to 5 kb inverted repeats. The repeat-associated centromeres of C. tropicalis all share a high degree of sequence conservation with each other and are strikingly diverged from the unique and mostly non-repetitive centromeres of related Candida species—Candida albicans, Candida dubliniensis, and Candida lusitaniae. Using a plasmid-based assay, we further demonstrate that pericentric inverted repeats and the underlying DNA sequence provide a structural determinant in CENP-A recruitment in C. tropicalis, as opposed to epigenetically regulated CENP-A loading at centromeres in C. albicans. Thus, the centromere structure and its influence on de novo CENP-A recruitment has been significantly rewired in closely related Candida species. Strikingly, the centromere structural properties along with role of pericentric repeats in de novo CENP-A loading in C. tropicalis are more reminiscent to those of the distantly related fission yeast Schizosaccharomyces pombe. Taken together, we demonstrate, for the first time, fission yeast-like repeat-associated centromeres in an ascomycetous budding yeast.

Bambusa balcooa is an economically important, multipurpose bamboo species, decidedly used in construction industry. Availability of natural bamboo is depleting very rapidly due to accelerated deforestation and its unrestrained use. The large number and timely supply of saplings are the need of the hour for the restoration of bamboo stands. Micropropagation, being the potent alternative for season independent rapid regeneration, is restricted in bamboo because of endophytic contamination. An in vitro attempt has been taken to overcome the endophytic contamination by using broad spectrum antibiotics as surface sterilant as well as a media component. Ampicillin sodium salt (5 mg/ml for 30 min) as a surface sterilant was found as the best treatment for high bud breaking (80%) coupled with high branching and low contamination (20%) but it was found ineffective to control the contamination during multiplication stage. Then, two endophytes were isolated and minimum inhibitory concentration was determined through antibiotic susceptibility test for successful eradication at multiplication stage. Finally, contamination free cultures were obtained when streptocycline (100 μg/ml) and gentamicin sulphate (75 μg/ml) were added into the medium. The two isolated endophytes, BB1 and BB2, were identified through 16S rDNA techniques and NCBI-BLAST algorithm with 99% sequence similarity with those of Janibacter sp. (KX423734) and Serratia marcescens strain (KX423735). To our knowledge, this is the first report for B. balcooa where antibiotics were used as surface sterilant as well as medium component, to control endophytic bacterial contaminants, followed by their identification.

Chromatin immunoprecipitation (ChIP) is a widely used technique which can determine the in vivo association of a specific protein on a particular DNA locus in the genome. In this method cross-linked chromatin is sheared and immunoprecipitated with antibodies raised against a target protein of interest. The end result of this process is the enrichment of DNA fragments associated with the desired protein. Thus, interactions between proteins and genomic loci in cellular context can be determined by this technique. Here, we are describing a ChIP protocol that is optimized for Candida albicans. The protocol requires 4-5 days for completion of the assay and has been used to produce robust ChIP results for diverse proteins in this organism and its related species including Candida dubliniensis and Candida tropicalis.

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India's oldest documented manure, most commonly referred to as Kunapajala, has a long history of over 1,000 years in crop cultivation. Kunapajala is primarily an in-situ decomposition technology of animal waste and can potentially provide an eco-friendly pipeline for recycling bio-waste into essential plant nutrients. This traditional animal manure, in addition, also contains dairy excreta (e.g., feces and urine), dairy products (e.g., milk and ghee), natural resources (e.g., honey), broken seeds or grains, and their non-edible by-product waste. Here, we aimed to assess the waste recycling and plant biostimulant potential of Kunapajala prepared from livestock (e.g., Black Bengal goats) or fish (e.g., Bombay duck) post-processed wastes over different decomposition periods, e.g., (0, 30, 60, and 90-days). In this study, an in-situ quantification of livestock- (lKPJ) and fish-based Kunapajala (fKPJ) reveals a dynamic landscape of essential plant primary nutrients, e.g., (0.70 > NH4-N < 3.40 g•L−1), (100.00 > P2O5 < 620.00 mg•L−1), and (175.00 > K2O < 340.00 mg•L−1), including other physico-chemical attributes of Kunapajala. Using correlation statistics, we find that the plant-available nutrient content of Kunapajala depicts a significant (p < 0.0001) transformation over decomposition along with microbial dynamics, abundance, and diversities, delineating a microbial interface to animal waste decomposition and plant growth promotion. Importantly, this study also reports the indole 3-acetic acid (IAA) content (40.00 > IAA < 135.00 mg•L−1) in Kunapajala. Furthermore, the bacterial screening based on plant growth-promoting traits and their functional analyses elucidate the mechanism of the plant biostimulant potential of Kunapajala. This assay finally reports two best-performing plant growth-promoting bacteria (e.g., Pseudomonas chlororaphis and Bacillus subtilis) by the 16S ribotyping method. In support, in-planta experiments have demonstrated, in detail, the bio-stimulative effects of Kunapajala, including these two bacterial isolates alone or in combination, on seed germination, root-shoot length, and other important agronomic, physio-biochemical traits in rice. Together, our findings establish that Kunapajala can be recommended as a source of plant biostimulant to improve crop quality traits in rice. Overall, this work highlights Kunapajala, for the first time, as a promising low-cost microbial technology that can serve a dual function of animal waste recycling and plant nutrient recovery to promote sustainable intensification in agroecosystems.

Beejamrit is an ancient organic formulation commonly used as a seed treatment in organic and natural farming in India. This low-cost input is primarily composed of cow dung, cow urine, and forest soil, which is often supplemented with limestone. In organic agriculture, it is a traditional practice among the farming community to incubate the seeds in Beejamrit prepared overnight before sowing in the field. However, a lack of substantial scientific evidence has been a major limitation to promote this farmers' innovative practice. On the other hand, growing data suggests that cow-based formulations are mostly enriched with microbial sources, including several plant growth-promoting rhizobacteria that are capable of synthesizing plant growth regulators. However, the microbiological properties of Beejamrit and their temporal changes over different periods of decomposition are largely unexplored. In this study, we aim to analyze the decomposition rate of Beejamrit over 7-consecutive days of incubation. This study further elucidates the microbial niche and their dynamics in Beejamrit, including the plant-beneficial bacteria. It was observed that the population of plant-beneficial bacteria such as the free-living nitrogen fixers and the phosphate solubilizers proliferates progressively up to 4-days of incubation. Similarly, the indolic compounds, including indole acetic acid, were enriched to the highest concentration in Beejamrit after 4-days of decomposition. Together, our analyses establish that Beejamrit provides a dynamic, microbe-based metabolic network and acts as a plant growth regulator. In conclusion, this study recommends the Beejamrit preparation after 4-days of incubation in the seed treatment and foliar spray to receive its optimum benefits.