Chromosome-level assembly of the Phytophthora agathidicida genome reveals adaptation in effector gene families.

Phytophthora species are notorious plant pathogens, with some causing devastating tree diseases that threaten the survival of their host species. One such example is Phytophthora agathidicida, the causal agent of kauri dieback - a root and trunk rot disease that kills the ancient, iconic and culturally significant tree species, Agathis australis (New Zealand kauri). A deeper understanding of how Phytophthora pathogens infect their hosts and cause disease is critical for the development of effective treatments. Such an understanding can be gained by interrogating pathogen genomes for effector genes, which are involved in virulence or pathogenicity. Although genome sequencing has become more affordable, the complete assembly of Phytophthora genomes has been problematic, particularly for those with a high abundance of repetitive sequences. Therefore, effector genes located in repetitive regions could be truncated or missed in a fragmented genome assembly. Using a combination of long-read PacBio sequences, chromatin conformation capture (Hi-C) and Illumina short reads, we assembled the P. agathidicida genome into ten complete chromosomes, with a genome size of 57 Mb including 34% repeats. This is the first Phytophthora genome assembled to chromosome level and it reveals a high level of syntenic conservation with the complete genome of Peronospora effusa, the only other completely assembled genome sequence of an oomycete. All P. agathidicida chromosomes have clearly defined centromeres and contain candidate effector genes such as RXLRs and CRNs, but in different proportions, reflecting the presence of gene family clusters. Candidate effector genes are predominantly found in gene-poor, repeat-rich regions of the genome, and in some cases showed a high degree of duplication. Analysis of candidate RXLR effector genes that occur in multicopy gene families indicated half of them were not expressed in planta. Candidate CRN effector gene families showed evidence of transposon-mediated recombination leading to new combinations of protein domains, both within and between chromosomes. Further analysis of this complete genome assembly will help inform new methods of disease control against P. agathidicida and other Phytophthora species, ultimately helping decipher how Phytophthora pathogens have evolved to shape their effector repertoires and how they might adapt in the future.

Effect of RNA silencing suppression activity of chrysanthemum virus B p12 protein on small RNA species.

Chrysanthemum virus B encodes a multifunctional p12 protein that acts as a transcriptional activator in the nucleus and as a suppressor of RNA silencing in the cytoplasm. Here, we investigated the impact of p12 on accumulation of major classes of small RNAs (sRNAs). The results show dramatic changes in the sRNA profiles characterised by an overall reduction in sRNA accumulation, changes in the pattern of size distribution of canonical siRNAs and in the ratio between sense and antisense strands, lower abundance of siRNAs with a U residue at the 5'-terminus, and changes in the expression of certain miRNAs, most of which were downregulated.

A Tripartite Interaction among the Basidiomycete Rhodotorula mucilaginosa, N2-Fixing Endobacteria, and Rice Improves Plant Nitrogen Nutrition.

Nitrogen (N) limits crop yield, and improvement of N nutrition remains a key goal for crop research; one approach to improve N nutrition is identifying plant-interacting, N2-fixing microbes. Rhodotorula mucilaginosa JGTA-S1 is a basidiomycetous yeast endophyte of narrowleaf cattail (Typha angustifolia). JGTA-S1 could not convert nitrate or nitrite to ammonium but harbors diazotrophic (N2-fixing) endobacteria (Pseudomonas stutzeri) that allow JGTA-S1 to fix N2 and grow in a N-free environment; moreover, P. stutzeri dinitrogen reductase was transcribed in JGTA-S1 even under adequate N. Endobacteria-deficient JGTA-S1 had reduced fitness, which was restored by reintroducing P. stutzeri JGTA-S1 colonizes rice (Oryza sativa), significantly improving its growth, N content, and relative N-use efficiency. Endofungal P. stutzeri plays a significant role in increasing the biomass and ammonium content of rice treated with JGTA-S1; also, JGTA-S1 has better N2-fixing ability than free-living P. stutzeri and provides fixed N to the plant. Genes involved in N metabolism, N transporters, and NODULE INCEPTION-like transcription factors were upregulated in rice roots within 24 h of JGTA-S1 treatment. In association with rice, JGTA-S1 has a filamentous phase and P. stutzeri only penetrated filamentous JGTA-S1. Together, these results demonstrate an interkingdom interaction that improves rice N nutrition.

Genome/transcriptome collection of plethora of economically important, previously unexplored organisms from India and abroad.

Genome and transcriptome sequencing data are extremely useful resources for researchers in carrying out biological experiments that involves cloning and characterizing genes. We are presenting here genome sequence data from different clades of life including photosynthetic prokaryotes; oomycetes pathogens; probiotic bacteria; endophytic yeasts and filamentous fungus and pathogenic protozoa Leishmania donovani. In addition, we are also presenting paired control and treated stress response transcriptomes of Cyanobacteria growing in extreme conditions. The Cyanobacterial species that are included in this dataset were isolated from extreme conditions including desiccated monuments, hot springs and saline archipelagos. The probiotic Lactobacillus paracasei was isolated from Indian sub-continent. The Kala azar causing protozoan Leishmania donovani, whose early infectious stage is also included in this dataset. The endophyte Arthrinium malaysianum was isolated as a contaminant has significant bio-remediation property. Our collaborators have isolated endophyte Rhodotorula mucilaginosa JGTA1 from Jaduguda mines, West Bengal, India infested with Uranium. Our collaborators have isolated a heterozygous diploid oomycetes pathogen, Phytophthora ramorum causing sudden oak death in CA, USA coast is also part of the data. These dataset presents a unique heterogeneous collection from various sources that are analyzed using "Genome Annotator Light (GAL): A Docker-based package for genome analysis and visualization" (Panda et al., 2019) and are presented in a web site automatically created by GAL at http://www.eumicrobedb.org/cglab.

A unique life-strategy of an endophytic yeast Rhodotorula mucilaginosa JGTA-S1-a comparative genomics viewpoint.

Endophytic yeasts of genus Rhodotorula are gaining importance for their ability to improve plant growth. The nature of their interaction with plants, however, remains unknown. Rhodotorula mucilaginosa JGTA-S1 was isolated as an endophyte of Typha angustifolia and promoted growth in the host. To investigate the life-strategy of the yeast from a genomics perspective, we used Illumina and Oxford Nanopore reads to generate a high-quality annotated draft assembly of JGTA-S1 and compared its genome to three other Rhodotorula yeasts and the close relative Rhodosporidium toruloides. JGTA-S1 is a haploid yeast possessing several genes potentially facilitating its endophytic lifestyle such as those responsible for solubilizing phosphate and producing phytohormones. An intact mating-locus in JGTA-S1 raised the possibility of a yet unknown sexual reproductive cycle in Rhodotorula yeasts. Additionally, JGTA-S1 had functional anti-freezing genes and was also unique in lacking a functional nitrate-assimilation pathway-a feature that is associated with obligate biotrophs. Nitrogen-fixing endobacteria were found within JGTA-S1 that may circumvent this defective N-metabolism. JGTA-S1 genome data coupled with experimental evidence give us an insight into the nature of its beneficial interaction with plants.

Genome Sequence Resource for the Oomycete Taro Pathogen Phytophthora colocasiae.

Phytophthora colocasiae is a phytopathogenic oomycete that causes leaf blight and corm rot on taro (Colocasia esculenta), an important staple crop in the tropics. The impact of P. colocasiae is a serious concern for food security in Asian and Oceanic regions. Vietnamese strain 7290 of P. colocasiae was sequenced (Illumina) to assemble a draft genome of 56.6 Mb, comprised of 19,853 scaffolds and 19,984 predicted protein-coding genes. As in other Phytophthora species, P. colocasiae possesses numerous pathogenicity-related genes, such as the RxLR class of effectors. This draft genome sequence of P. colocasiae provides a resource to underpin the first steps in determining the molecular mechanisms of disease development in this pathosystem.

EumicrobeDBLite: a lightweight genomic resource and analytic platform for draft oomycete genomes.

We have developed EumicrobeDBLite-a lightweight comprehensive genome resource and sequence analysis platform for oomycete organisms. EumicrobeDBLite is a successor of the VBI Microbial Database (VMD) that was built using the Genome Unified Schema (GUS). In this version, GUS has been greatly simplified with the removal of many obsolete modules and the redesign of others to incorporate contemporary data. Several dependences, such as perl object layers used for data loading in VMD, have been replaced with independent lightweight scripts. EumicrobeDBLite now runs on a powerful annotation engine developed at our laboratory, called 'Genome Annotator Lite'. Currently, this database has 26 publicly available genomes and 10 expressed sequence tag (EST) datasets of oomycete organisms. The browser page has dynamic tracks presenting comparative genomics analyses, coding and non-coding data, tRNA genes, repeats and EST alignments. In addition, we have defined 44 777 core conserved proteins from 12 oomycete organisms which form 2974 clusters. Synteny viewing is enabled by the incorporation of the Genome Synteny Viewer (GSV) tool. The user interface has undergone major changes for ease of browsing. Queryable comparative genomics information, conserved orthologous genes and pathways are among the new key features updated in this database. The browser has been upgraded to enable user upload of GFF files for quick view of genome annotation comparisons. The toolkit page integrates the EMBOSS package and has a gene prediction tool. Annotations for the organisms are updated once every 6 months to ensure quality. The database resource is available at www.eumicrobedb.org.

Draft Genome Sequence of the Terrestrial Cyanobacterium Scytonema millei VB511283, Isolated from Eastern India.

We report here the draft genome sequence of Scytonema millei VB511283, a cyanobacterium isolated from biofilms on the exterior of stone monuments in Santiniketan, eastern India. The draft genome is 11,627,246 bp long (11.63 Mb), with 118 scaffolds. About 9,011 protein-coding genes, 117 tRNAs, and 12 rRNAs are predicted from this assembly.

Draft Genome Sequence of Cyanobacterium Hassallia byssoidea Strain VB512170, Isolated from Monuments in India.

The draft genome assembly of Hassallia byssoidea strain VB512170 with a genome size of ~13 Mb and 10,183 protein-coding genes in 62 scaffolds is reported here for the first time. This is a terrestrial hydrophobic cyanobacterium isolated from monuments in India. We report several copies of luciferase and antibiotic genes in this organism.

Draft Genome Sequence of Filamentous Marine Cyanobacterium Lyngbya confervoides Strain BDU141951.

Lyngbya confervoides strain BDU141951 is a fast-growing, unicellular, marine, nonheterocystous cyanobacterium forming long unbranched filaments inside sheaths. Here, we report the draft genome assembly of Lyngbya confervoides BDU141951 for the first time. The genome size is 8,799,693 bp and has 6,093 putative protein-coding genes assembled into 298 scaffolds.

Draft Genome Sequence of Tolypothrix boutellei Strain VB521301.

We report here the draft genome sequence of the filamentous nitrogen-fixing cyanobacterium Tolypothrix boutellei strain VB521301. The organism is lipid rich and hydrophobic and produces polyunsaturated fatty acids which can be harnessed for industrial purpose. The draft genome sequence assembled into 11,572,263 bp with 70 scaffolds and 7,777 protein coding genes.

Diverse broad-host-range plasmids from freshwater carry few accessory genes.

Broad-host-range self-transferable plasmids are known to facilitate bacterial adaptation by spreading genes between phylogenetically distinct hosts. These plasmids typically have a conserved backbone region and a variable accessory region that encodes host-beneficial traits. We do not know, however, how well plasmids that do not encode accessory functions can survive in nature. The goal of this study was to characterize the backbone and accessory gene content of plasmids that were captured from freshwater sources without selecting for a particular phenotype or cultivating their host. To do this, triparental matings were used such that the only required phenotype was the plasmid's ability to mobilize a nonconjugative plasmid. Based on complete genome sequences of 10 plasmids, only 5 carried identifiable accessory gene regions, and none carried antibiotic resistance genes. The plasmids belong to four known incompatibility groups (IncN, IncP-1, IncU, and IncW) and two potentially new groups. Eight of the plasmids were shown to have a broad host range, being able to transfer into alpha-, beta-, and gammaproteobacteria. Because of the absence of antibiotic resistance genes, we resampled one of the sites and compared the proportion of captured plasmids that conferred antibiotic resistance to their hosts with the proportion of such plasmids captured from the effluent of a local wastewater treatment plant. Few of the captured plasmids from either site encoded antibiotic resistance. A high diversity of plasmids that encode no or unknown accessory functions is thus readily found in freshwater habitats. The question remains how the plasmids persist in these microbial communities.

Inferring the evolutionary history of IncP-1 plasmids despite incongruence among backbone gene trees.

Plasmids of the incompatibility group IncP-1 can transfer and replicate in many genera of the Proteobacteria. They are composed of backbone genes that encode a variety of essential functions and accessory genes that have implications for human health and environmental remediation. Although it is well understood that the accessory genes are transferred horizontally between plasmids, recent studies have also provided examples of recombination in the backbone genes of IncP-1 plasmids. As a consequence, phylogeny estimation based on backbone genes is expected to produce conflicting gene tree topologies. The main goal of this study was therefore to infer the evolutionary history of IncP-1 plasmids in the presence of both vertical and horizontal gene transfer. This was achieved by quantifying the incongruence among gene trees and attributing it to known causes such as 1) phylogenetic uncertainty, 2) coalescent stochasticity, and 3) horizontal inheritance. Topologies of gene trees exhibited more incongruence than could be attributed to phylogenetic uncertainty alone. Species-tree estimation using a Bayesian framework that takes coalescent stochasticity into account was well supported, but it differed slightly from the maximum-likelihood tree estimated by concatenation of backbone genes. After removal of the gene that demonstrated a signal of intergroup recombination, the concatenated tree was congruent with the species-tree estimate, which itself was robust to inclusion/exclusion of the recombinant gene. Thus, in spite of horizontal gene exchange both within and among IncP-1 subgroups, the backbone genome of these IncP-1 plasmids retains a detectable vertical evolutionary history.

Broad-host-range plasmids from agricultural soils have IncP-1 backbones with diverse accessory genes.

Broad-host-range plasmids are known to spread genes between distinct phylogenetic groups of bacteria. These genes often code for resistances to antibiotics and heavy metals or degradation of pollutants. Although some broad-host-range plasmids have been extensively studied, their evolutionary history and genetic diversity remain largely unknown. The goal of this study was to analyze and compare the genomes of 12 broad-host-range plasmids that were previously isolated from Norwegian soils by exogenous plasmid isolation and that encode mercury resistance. Complete nucleotide sequencing followed by phylogenetic analyses based on the relaxase gene traI showed that all the plasmids belong to one of two subgroups (ß and ε) of the well-studied incompatibility group IncP-1. A diverse array of accessory genes was found to be involved in resistance to antimicrobials (streptomycin, spectinomycin, and sulfonamides), degradation of herbicides (2,4-dichlorophenoxyacetic acid and 2,4-dichlorophenoxypropionic acid), and a putative new catabolic pathway. Intramolecular transposition of insertion sequences followed by deletion was found to contribute to the diversity of some of these plasmids. The previous observation that the insertion sites of a Tn501-related element are identical in four IncP-1ß plasmids (pJP4, pB10, R906, and R772) was further extended to three more IncP-1ß plasmids (pAKD15, pAKD18, and pAKD29). We proposed a hypothesis for the evolution of these Tn501-bearing IncP-1ß plasmids that predicts recent diversification followed by worldwide spread. Our study increases the available collection of complete IncP-1 plasmid genome sequences by 50% and will aid future studies to enhance our understanding of the evolution and function of this important plasmid family.

Comparative genomics of pAKD4, the prototype IncP-1delta plasmid with a complete backbone.

Plasmids of the incompatibility group IncP-1 are important agents of horizontal gene transfer and contribute to the spread of antibiotic resistance and xenobiotic degradation within bacterial communities. Even though some prototype plasmids have been studied in much detail, the diversity of this plasmid group was still greatly underestimated until recently, as only two of the five currently known divergent sub-groups had been described. To further improve our insight into the diversity and evolutionary history of this family of broad-host-range plasmids, we compared the complete nucleotide sequence of a new IncP-1delta plasmid pAKD4 to the genomes of other IncP-1 plasmids. Plasmid pAKD4 was previously isolated by exogenous plasmid isolation from an agricultural soil in Norway. Its 56,803bp nucleotide sequence shows high similarity in gene sequence and gene order to both plasmids pEST4011 and pIJB1, the only other IncP-1delta plasmids sequenced so far. While all three plasmids have a typical IncP-1 backbone comprising replication, transfer, and stable inheritance/control genes, the low sequence similarity in some regions and presence/absence of some backbone genes compared to other IncP-1 plasmids cluster them in a divergent sub-group. Therefore this study validates the presence of a real IncP-1delta clade with multiple plasmids. Moreover, since both pEST4011 and pIJB1 are missing a portion of their transfer genes, pAKD4 represents the first completely sequenced self-transferable plasmid with a complete IncP-1delta backbone. We therefore propose it to be the prototype IncP-1delta plasmid.