Biology subjects

Triplett, L. R.

Publications and source records attributed to Triplett, L. R..

7 recordsLinked to original sources

bioRxiv2026

A qPCR method facilitates study of absolute abundance, ecology, and inoculation fate of ciliate predators on the leaf surface

Predatory protists are important in shaping terrestrial microbial ecosystems, but their roles in the phyllosphere, or the communities on aerial plant surfaces, are poorly understood. Previous work found that the order Colpodida dominated heterotrophic protist communities in the phyllosphere. While most protists were sporadically present, a few Colpodida variants were prevalent and abundant, indicating that these variants may represent species adapted to the phyllosphere. To identify these organisms, we cultured colpodids from field-collected tomato leaves and performed phylogenetic analysis of the 18S rRNA gene. Five of nine independent isolates matched the most prevalent Colpodida variant previously identified as leaf-enriched through amplicon sequencing, and these isolates comprised a novel clade of Paracolpoda steinii. When compared to a maize root isolate of Colpoda inflata, an abundant rhizosphere ciliate, a P. steinii isolate was similar in size and growth yield on E. coli, but grew to higher yields and formed large cyst clusters when incubated with model phyllosphere bacteria prey Erwinia and Pseudomonas. We developed and validated quantitative PCR (qPCR) methods for detection and cell abundance estimation of the P. steinii phyllosphere clade, C. inflata, and the order Colpodida in environmental samples. In inoculated greenhouse plants, qPCR-estimated protist populations matched measured inoculum levels, and protist inoculum was still detectable after five days. In an uninoculated tomato field, P. steinii was detected on all plants, with greatest abundances observed in lower leaves and after a rain event. P. steinii comprised up to 18.7% of total leaf Colpodida populations, which were estimated at up to [~]1400 organisms per gram of fresh weight. The findings demonstrate that Colpodida communities are consistently present on tomato leaves, dynamically affected by the abiotic environment, and include significant populations of P. steinii. We propose that the P. steinii isolates and qPCR tools presented can be used as a model system to investigate colonization and distribution patterns, biotic interactions, genetic adaptations, and agricultural applications of leaf predation.

microbiology↗

bioRxiv2025

Neuromorpha vorax: a previously unculturable cosmopolitan protist with an unexpectedly complex life-cycle belonging to Glissomonadida Clade-U/Group-TE

Soil protists are increasingly recognized as common members of complex communities that associate with plant root systems, though their contributions to these communities and to the plant host remain obscure. Members Clade-U/Group-TE, are cosmopolitan soil protists and are often among the most abundant protists associated with plant roots. Here we describe the isolation, culturing and characterization of Neuromorpha vorax, a member of the previously uncultured Clade-U/Group-TE branch of the order Glissomonadida. N. vorax grew readily when provided bacteriophage lysate as a food source. This allowed us to grow large numbers of the organisms from single-cell isolates and provided ideal conditions for following transitions from one morphology to another. N. vorax, like most Glissomonads, has a small, flagellated gliding form, but it also displays a wide range of other morphologies including: a crawling form; small and large trophozoites with multiple, long filopodia; small and large resting cysts; clusters of large dividing cells and it displayed cannibalistic feeding behaviors. Given the small size of most Glissomonads, it may be that other members of this important group, known from environmental surveys, but currently uncultured, might also be readily grown on bacteriophage lysates. In addition, given an abundant food source and clear viewing by microscopy, Glissomonads and other small protists may be found to have life cycles and behaviors that are more complex than is currently appreciated. ImportanceProtists from the Clade-U/Group-TE cluster of Glissomonads are wide-spread and abundant colonizers plant roots. Despite being known for over 30 years, they have remained uncultured. We show these protists can be easily cultured using an unusual food source, viral lysates of bacteria. This culturing method allows growth of high numbers of these organisms and reveal thatthey have an unexpectedly complex lifecycle that includes community feeding and cannibalism. Some other, currently unculturable, protists can perhaps be grown with these methods and many of these may also show unexpectedly complex lifecycles. The growth of eukaryotes on virus lysates raises the possibility that viruses in soil may directly contribute to the growth (and not just death) of eukaryotes in soil and root-associated communities.

microbiology↗

bioRxiv2025

Rhizosphere-colonizing bacteria persist in the protist microbiome

Soils contain diverse predatory protists that affect the abundance and behavior of rhizosphere bacteria, including bacteria that may benefit plant health. Protists also harbor their own bacterial microbiomes, including transient and extracellular associates, but it is not known whether the protist microbiome affects the plant rhizosphere. To address this question, we profiled the bacterial microbiomes of eight evolutionarily diverse rhizosphere protist isolates after two years of continuous laboratory culture. We then compared the protist culture microbiomes to maize rhizosphere communities six weeks after protist inoculation. Introduction of protists enriched 13 protist-associated bacterial amplicon sequence variants (ASVs) in the rhizosphere, which comprised [~]10% of the rhizosphere bacterial community. Additional bacterial ASVs ranked highly in abundance in both rhizosphere and protist microbiomes; together, a median 47% of the protist microbiome was enriched or abundant in the rhizosphere. Inoculation with some of the protist cultures positively affected root biomass traits, but a protist mixture had no effect, indicating that the impact of protist holobionts on plant growth is context-dependent. Isolates of protist-associated bacteria had both positive and negative effects on protist growth in culture, suggesting that the bacteria use multiple strategies to survive in proximity to predators. This study demonstrates that even after prolonged laboratory culture, evolutionarily diverse rhizosphere protists host bacterial microbiomes dominated by plant-colonizing bacteria that impact the rhizosphere microbiome after inoculation. The findings suggest that protists may contribute to the rhizosphere as part of the soil microbial seedbank, and identify bacterial groups that may be important to the plant-protist interaction. ImportanceUnderstanding the impact of predatory protists on the plant microbiome will be essential to deploy protists in sustainable agriculture. This study shows that eight rhizosphere protist isolates hosted diverse and distinct bacterial communities, and that a large proportion of these bacteria could be found colonizing the maize root environment long after protists were introduced. This study demonstrates that maize rhizosphere bacteria can persist for years in the protist microbiome, indicating that extremely diverse eukaryotes could help select and maintain rhizosphere bacteria in the absence of the plant.

microbiology↗

bioRxiv2021

A dual sequence and culture-based survey of maize rhizosphere protists reveals dominant,plant-enriched, and culturable community members

Protists play important roles in shaping the microbial community of the rhizosphere. However, there is still a limited understanding of how plants shape the protist community, and how well protist isolate collections might represent rhizosphere protist composition and function in downstream studies. We sought to determine whether maize roots select for a distinct protist community in the field, and whether the common or dominant members of that community are readily culturable using standard protist isolation methods. We sequenced 18S and 16S rRNA genes from the rhizospheres of maize grown in two sites, and isolated 103 protists into culture from the same roots. While field site had the greatest effect, rhizospheres in both sites had distinct protist composition from the bulk soils, and certain taxa were enriched in both sites. Enriched taxa were correlated to bacterial abundance patterns. The isolated protists represented six supergroups, and the majority corresponded to taxa found in the sequencing survey. Twenty-six isolates matched eight of the 89 core rhizosphere taxa. This study demonstrates that maize roots select for a distinct protist community, but also illustrate the potential challenges in understanding the function of the dominant protist groups in the rhizosphere. Originality-Significance StatementThis is the first study comparing cultivation-dependent and independent methods for studying the protist community of plant roots, and the first untargeted analysis of the maize rhizospheres effect on protist communities. We show that maize in different sites select for distinct communities and overlapping enriched taxa, but that isolating the most important plant-associated protists may be a challenge for researchers.

microbiology↗

bioRxiv2020

Validation of a PNA clamping method for reducing host DNA amplification and increasing eukaryotic diversity in rhizosphere microbiome studies

Protists and microscopic animals are important but poorly understood determinants of plant health. Plant-associated eukaryotes could be surveyed by high-throughput sequencing of 18S ribosomal RNA (rRNA) genes, but the abundance of plant DNA in rhizosphere samples makes 18S rRNA gene amplification with universal primers unfeasible. Here we applied a pipeline to generate peptide nucleic acid (PNA) clamps to suppress the amplification of plant host DNA during 18S rRNA gene library preparation. We designed a PNA clamp, PoacV9_01, specific to the V9 region of the 18S rRNA gene for plants in the family Poaceae. PoacV9_01 suppressed the amplification of five species of grain crops in quantitative PCR reactions. In an 18S rRNA gene sequencing survey of the rhizosphere of maize, PoacV9_01 reduced the relative abundance of plant reads from 65% to 0.6%, while drastically increasing the number and diversity of animal, fungal, and protist reads detected. Thus, PoacV9_01 can be used to facilitate the study of eukaryotes present in grass phytobiomes. In addition, the pipeline developed here can be used to develop PNA clamps that target other plant species.

microbiology↗

bioRxiv2019

Genome assembly and characterization of a complex zfBED-NLR gene-containing disease resistance locus in Carolina Gold Select rice with Nanopore sequencing

BackgroundLong-read sequencing facilitates assembly of complex genomic regions. In plants, loci containing nucleotide-binding, leucine-rich repeat (NLR) disease resistance genes are an important example of such regions. NLR genes make up one of the largest gene families in plants and are often clustered, evolving via duplication, contraction, and transposition. We recently mapped the Xo1 locus for resistance to bacterial blight and bacterial leaf streak, found in the American heirloom rice variety Carolina Gold Select, to a region that in the Nipponbare reference genome is rich in NLR genes.\n\nResultsToward identification of the Xo1 gene, we combined Nanopore and Illumina reads to generate a high-quality genome assembly for Carolina Gold Select. We identified 529 full or partial NLR genes and discovered, relative to the reference, an expansion of NLR genes at the Xo1 locus. One NLR gene at Xo1 has high sequence similarity to the cloned, functionally similar Xa1 gene. Both harbor an integrated zfBED domain and near-identical, tandem, C-terminal repeats. Across diverse Oryzeae, we identified two sub-clades of such NLR genes, varying in the presence of the zfBED domain and the number of repeats.\n\nConclusionsWhole genome sequencing combining Nanopore and Illumina reads effectively resolves NLR gene loci, providing context as well as content. Our identification of an Xo1 candidate is an important step toward mechanistic characterization, including the role(s) of the zfBED domain. Further, the Carolina Gold Select genome assembly will facilitate identification and exploitation of other useful traits in this historically important rice variety.

plant biology↗

bioRxiv2019

Cell-length heterogeneity: a population-level solution to growth/virulence trade-offs in the plant pathogen Dickeya dadantii

Necrotrophic plant pathogens acquire nutrients from dead plant cells, which requires the disintegration of the plant cell wall and tissue structures by the pathogen. Infected plants lose tissue integrity and functional immunity as a result, exposing the nutrient rich, decayed tissues to the environment. One challenge for the necrotrophs to successfully cause secondary infection (infection spread from an initially infected plant to the nearby uninfected plants) is to effectively utilize nutrients released from hosts towards building up a large population before other saprophytes come. In this study, we observed that the necrotrophic pathogen Dickeya dadantii exhibited heterogeneity in bacterial cell length in an isogenic population during infection of potato tuber. While some cells were regular rod-shape (<10m), the rest elongated into filamentous cells (>10m). Short cells tended to occur at the interface of healthy and diseased tissues, during the early stage of infection when active attacking and killing is occurring, while filamentous cells tended to form when large amount of nutrients were released at a later stage of infection. Short cells expressed all necessary virulence factors and motility, whereas filamentous cells did not engage in virulence, were non-mobile and more sensitive to environmental stress. However, compared to the short cells, the filamentous cells displayed elevated metabolism and faster growth, which may benefit the pathogens to build up a large population necessary for the secondary infection. The segregation of the two subpopulations was dependent on differential expression of the alarmone guanosine tetraphosphate (ppGpp). When exposed to fresh tuber tissues or freestanding water, filamentous cells quickly transformed to short virulent cells. The pathogen adaptation of cell length heterogeneity identified in this study presents a model for how some necrotrophs balance virulence and vegetative growth to maximize fitness during infection.

microbiology↗