Coevolution Of Host Research Articles

Is Color Related to Parasite Load in a Sexually Dichromatic Neotropical Toad?

Research on anuran parasites often focuses on the description and identification of parasites and demonstrating that parasitic infection can have deleterious effects on the health of amphibians. There is comparatively little research on how parasitism influences evolutionary dynamics such as sexual selection. Here, we explore anuran blood parasites with a focus on parasite-mediated sexual selection. The Hamilton–Zuk hypothesis posits that parasite–host coevolution drives sexual ornamentation of hosts, and that the degree of ornament expression honestly signals heritable parasite resistance. Although the Hamilton–Zuk hypothesis has received support in a number of animal taxa, it has received little attention in amphibians. Neotropical Yellow Toads (Incilius luetkenii) are sexually dichromatic toads with an unusual sexual ornament: males turn from cryptic brown to bright lemon yellow for a very short period during the brief breeding season. In this study, we identify blood parasites in male Neotropical Yellow Toads, and we investigate one component of the Hamilton–Zuk hypothesis by comparing skin color and body condition to blood parasite levels. We found four types of parasites. Apicomplexa and frog erythrocytic virus were the most common, infecting 73% and 46% of toads, respectively, whereas Nematoda and bacteria were found in 27% and 36% of toads, respectively. We found negative relationships between parasites and male color: males infected with fewer Apicomplexa and Nematoda had higher yellow saturation, and males with fewer Nematoda had longer-wavelength hue. In contrast, we found one positive relationship between parasites and male color: males infected with higher levels of frog erythrocytic virus had higher brightness. Our results suggest that parasites may influence sexual traits in this species, but that the relationship is complex. This research contributes to our understanding of amphibian host–parasite coevolution by demonstrating a relationship between parasites and host coloration.

Human neutrophils murine neutrophils: Does it matter?

Human and murine neutrophils differ with respect to representation in blood, receptors, nuclear morphology, signaling pathways, granule proteins, NADPH oxidase regulation, magnitude of oxidant and hypochlorous acid production, and their repertoire of secreted molecules. These differences often matter and can undermine extrapolations from murine studies to clinical care, as illustrated by several failed therapeutic interventions based on mouse models. Likewise, coevolution of host and pathogen undercuts fidelity of murine models of neutrophil-predominant human infections. However, murine systems that accurately model the human condition can yield insights into human biology difficult to obtain otherwise. The challenge for investigators who employ murine systems is to distinguish models from pretenders and to know when the mouse provides biologically accurate insights. Testing with human neutrophils observations made in murine systems would provide a safeguard but is not always possible. At a minimum, studies that use exclusively murine neutrophils should have accurate titles supported by data and restrict conclusions to murine neutrophils and not encompass all neutrophils. For now, the integration of evidence from studies of neutrophil biology performed using valid murine models coupled with testing in vitro of human neutrophils combines the best of both approaches to elucidate the mysteries of human neutrophil biology.

Nuclear antiviral innate responses at the intersection of DNA sensing and DNA repair.

The coevolution of vertebrate and mammalian hosts with DNA viruses has driven the ability of host cells to distinguish viral from cellular DNA in the nucleus to induce intrinsic immune responses. Concomitant viral mechanisms have arisen to inhibit DNA sensing. At this virus-host interface, emerging evidence links cytokine responses and cellular homeostasis pathways, particularly the DNA damage response (DDR). Nuclear DNA sensors, such as the interferon (IFN)-γ inducible protein 16 (IFI16), functionally intersect with the DDR regulators ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK). Here, we discuss accumulating knowledge for the DDR-innate immunity signaling axis. Through the lens of this infection-driven signaling axis, we present host and viral molecular strategies acquired to regulate autoinflammation and antiviral responses.

Novel antiinflammatory biologics shaped by parasite–host coevolution

Parasitic helminth infections, while a major cause of neglected tropical disease burden, negatively correlate with the incidence of immune-mediated inflammatory diseases such as inflammatory bowel diseases (IBD). To evade expulsion, helminths have developed sophisticated mechanisms to regulate their host's immune responses. Controlled experimental human helminth infections have been assessed clinically for treating inflammatory conditions; however, such a radical therapeutic modality has challenges. An alternative approach is to harness the immunomodulatory properties within the worm's excretory-secretory (ES) complement, its secretome. Here, we report a biologics discovery and validation pipeline to generate and screen invivo a recombinant cell-free secretome library of helminth-derived immunomodulatory proteins. We successfully expressed 78 recombinant ES proteins from gastrointestinal hookworms and screened the crude invitro translation reactions for anti-IBD properties in a mouse model of acute colitis. After statistical filtering and ranking, 20 proteins conferred significant protection against various parameters of colitis. Lead candidates from distinct protein families, including annexins, transthyretins, nematode-specific retinol-binding proteins, and SCP/TAPS were identified. Representative proteins were produced in mammalian cells and further validated, including ex vivo suppression of inflammatory cytokine secretion by T cells from IBD patient colon biopsies. Proteins identified herein offer promise as novel, safe, and mechanistically differentiated biologics for treating the globally increasing burden of inflammatory diseases.

Detection of Cetacean Poxvirus in Peruvian Common Bottlenose Dolphins (Tursiops truncatus) Using a Pan-Poxvirus PCR.

Cetacean poxviruses (CePVs) cause ‘tattoo’ skin lesions in small and large cetaceans worldwide. Although the disease has been known for decades, genomic data for these poxviruses are very limited, with the exception of CePV-Tursiops aduncus, which was completely sequenced in 2020. Using a newly developed pan-pox real-time PCR system targeting a conserved nucleotide sequence located within the Monkeypox virus D6R gene, we rapidly detected the CePV genome in typical skin lesions collected from two Peruvian common bottlenose dolphins (Tursiops truncatus) by-caught off Peru in 1993. Phylogenetic analyses based on the sequencing of the DNA polymerase and DNA topoisomerase genes showed that the two viruses are very closely related to each other, although the dolphins they infected pertained to different ecotypes. The poxviruses described in this study belong to CePV-1, a heterogeneous clade that infects many species of dolphins (Delphinidae) and porpoises (Phocoenidae). Among this clade, the T. truncatus CePVs from Peru were more related to the viruses infecting Delphinidae than to those detected in Phocoenidae. This is the first time that CePVs were identified in free-ranging odontocetes from the Eastern Pacific, surprisingly in 30-year-old samples. These data further suggest a close and long-standing pathogen–host co-evolution, resulting in different lineages of CePVs.

Competition and coevolution drive the evolution and the diversification of CRISPR immunity.

The diversity of resistance challenges the ability of pathogens to spread and to exploit host populations. Yet, how this host diversity evolves over time remains unclear because it depends on the interplay between intraspecific competition among host genotypes and coevolution with pathogens. Here we study experimentally the effect of coevolving phage populations on the diversification of bacterial CRISPR immunity across space and time. We demonstrate that the negative-frequency-dependent selection generated by coevolution is a powerful force that maintains host resistance diversity and selects for new resistance mutations in the host. We also find that host evolution is driven by asymmetries in competitive abilities among different host genotypes. Even if the fittest host genotypes are targeted preferentially by the evolving phages, they often escape extinctions through the acquisition of new CRISPR immunity. Together, these fluctuating selective pressures maintain diversity, but not by preserving the pre-existing host composition. Instead, we repeatedly observe the introduction of new resistance genotypes stemming from the fittest hosts in each population. These results highlight the importance of competition on the transient dynamics of host-pathogen coevolution.

The avian gut microbiota: Diversity, influencing factors, and future directions.

The gut microbiota is viewed as the “second genome” of animals, sharing intricate relationships with their respective hosts. Because the gut microbial community and its diversity are affected by many intrinsic and extrinsic factors, studying intestinal microbes has become an important research topic. However, publications are dominated by studies on domestic or captive birds, while research on the composition and response mechanism of environmental changes in the gut microbiota of wild birds remains scarce. Therefore, it is important to understand the co-evolution of host and intestinal bacteria under natural conditions to elucidate the diversity, maintenance mechanisms, and functions of gut microbes in wild birds. Here, the existing knowledge of gut microbiota in captive and wild birds is summarized, along with previous studies on the composition and function, research methods employed, and factors influencing the avian gut microbial communities. Furthermore, research hotspots and directions were also discussed to identify the dynamics of the avian gut microbiota, aiming to contribute to studies of avian microbiology in the future.

Predation shifts coevolution toward higher host contact rate and parasite virulence.

Hosts can avoid parasites (and pathogens) by reducing social contact, but such isolation may carry costs, e.g. increased vulnerability to predators. Thus, many predator-host-parasite systems confront hosts with a trade-off between predation and parasitism. Parasites, meanwhile, evolve higher virulence in response to increased host sociality and consequently, increased multiple infections. How does predation shift coevolution of host behaviour and parasite virulence? What if predators are selective, i.e. predators disproportionately capture the sickest hosts? We answer these questions with an eco-coevolutionary model parametrized for a Trinidadian guppy-Gyrodactylus spp. system. Here, increased predation drives host coevolution of higher grouping, which selects for higher virulence. Additionally, higher predator selectivity drives the contact rate higher and virulence lower. Finally, we show how predation and selectivity can have very different impacts on host density and prevalence depending on whether hosts or parasites evolve, or both. For example, higher predator selectivity led to lower prevalence with no evolution or only parasite evolution but higher prevalence with host evolution or coevolution. These findings inform our understanding of diverse systems in which host behavioural responses to predation may lead to increased prevalence and virulence of parasites.

GenBank as a source to monitor and analyze Host-Microbiome data.

Microbiome datasets are often constrained by sequencing limitations. GenBank is the largest collection of publicly available DNA sequences, which is maintained by the National Center of Biotechnology Information (NCBI). The metadata of GenBank records are a largely understudied resource and may be uniquely leveraged to access the sum of prior studies focused on microbiome composition. Here, we developed a computational pipeline to analyze GenBank metadata, containing data on hosts, microorganisms and their place of origin. This work provides the first opportunity to leverage the totality of GenBank to shed light on compositional data practices that shape how microbiome datasets are formed as well as examine host-microbiome relationships. The collected dataset contains multiple kingdoms of microorganisms, consisting of bacteria, viruses, archaea, protozoa, fungi, and invertebrate parasites, and hosts of multiple taxonomical classes, including mammals, birds and fish. A human data subset of this dataset provides insights to gaps in current microbiome data collection, which is biased towards clinically relevant pathogens. Clustering and phylogenic analysis reveals the potential to use these data to model host taxonomy and evolution, revealing groupings formed by host diet, environment and coevolution. GenBank Host-Microbiome Pipeline is available at https://github.com/bcbi/genbank_holobiome. The GenBank loader is available at https://github.com/bcbi/genbank_loader. Supplementary data are available at Bioinformatics online.

Phage-host coevolution in natural populations.

Coevolution between bacteriophages (phages) and their bacterial hosts occurs through changes in resistance and counter-resistance mechanisms. To assess phage-host evolution in wild populations, we isolated 195 Vibrio crassostreae strains and 243 vibriophages during a 5-month time series from an oyster farm and combined these isolates with existing V. crassostreae and phage isolates. Cross-infection studies of 81,926 host-phage pairs delineated a modular network where phages are best at infecting co-occurring hosts, indicating local adaptation. Successful propagation of phage is restricted by the ability to adsorb to closely related bacteria and further constrained by strain-specific defence systems. These defences are highly diverse and predominantly located on mobile genetic elements, and multiple defences are active within a single genome. We further show that epigenetic and genomic modifications enable phage to adapt to bacterial defences and alter host range. Our findings reveal that the evolution of bacterial defences and phage counter-defences is underpinned by frequent genetic exchanges with, and between, mobile genetic elements.

Topological analysis of orchid-fungal endophyte interaction shows lack of phylogenetic preference

Fungal endophytes are known to play a vital role in perpetuation and survival of orchids. While orchid–mycorrhizal relationship has been studied in detail, dynamics of orchid–non-mycorrhizal associations are insufficiently explored. There is paucity of empirical evidence for host specificity, phylogenetic preference, co-evolution, and habitat selection amongst orchids and their non-mycorrhizal endophytes. We explored these aspects by studying the root associated non-mycorrhizal fungal assemblages from 11 epiphytic orchid species in two tropical communities located at Indo-Burma biodiversity hotspot. A total of 94 non-mycorrhizal fungal isolates were mined. Of these 55 isolates could be identified at species level, while 39 isolates were circumscribed at generic level using the ITS region. The core group fungal assemblages predominantly belonged to Ascomycota. The extent of mutualistic reliance was inferred by mapping the bipartite interaction in a phylogenetic framework. The analysis shows that the orchid–non-mycorrhizal associations are neither clade specific nor nested, rather they depend on the availability of suitable fungal taxon at the sites. The study also indicates the absence of phylogenetic preference between the partners. However, topology of the bipartite interaction network suggests that there has been a shift from specialised interaction in the basal clades, to generalised one in the later evolved clades.

Knockout of SlTOM1 and SlTOM3 results in differential resistance to tobamovirus in tomato.

During tobamovirus–host coevolution, tobamoviruses developed numerous interactions with host susceptibility factors and exploited these interactions for replication and movement. The plant‐encoded TOBAMOVIRUS MULTIPLICATION (TOM) susceptibility proteins interact with the tobamovirus replicase proteins and allow the formation of the viral replication complex. Here CRISPR/Cas9‐mediated mutagenesis allowed the exploration of the roles of SlTOM1a, SlTOM1b, and SlTOM3 in systemic tobamovirus infection of tomato. Knockouts of both SlTOM1a and SlTOM3 in sltom1a/sltom3 plants resulted in an asymptomatic response to the infection with recently emerged tomato brown rugose fruit virus (ToBRFV). In addition, an accumulation of ToBRFV RNA and coat protein (CP) in sltom1a/sltom3 mutant plants was 516‐ and 25‐fold lower, respectively, than in wild‐type (WT) plants at 12 days postinoculation. In marked contrast, sltom1a/sltom3 plants were susceptible to previously known tomato viruses, tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV), indicating that SlTOM1a and SlTOM3 are not essential for systemic infection of TMV and ToMV in tomato plants. Knockout of SlTOM1b alone did not contribute to ToBRFV and ToMV resistance. However, in triple mutants sltom1a/sltom3/sltom1b, ToMV accumulation was three‐fold lower than in WT plants, with no reduction in symptoms. These results indicate that SlTOM1a and SlTOM3 are essential for the replication of ToBRFV, but not for ToMV and TMV, which are associated with additional susceptibility proteins. Additionally, we showed that SlTOM1a and SlTOM3 positively regulate the tobamovirus susceptibility gene SlARL8a3. Moreover, we found that the SlTOM family is involved in the regulation of plant development.

Anaerobic ciliates as a model group for studying symbioses in oxygen-depleted environments.

Anaerobiosis has independently evolved in multiple lineages of ciliates, allowing them tocolonize a variety of anoxic and oxygen-depleted habitats. Anaerobic ciliates commonly form symbiotic relationships with various prokaryotes, including methanogenic archaea and members of several bacterial groups. The hypothesized functions ofthese ecto- and endosymbionts include the symbiont utilizing the ciliate's fermentative end products to increase the host's anaerobic metabolic efficiency, or the symbiont directly providing the host with energy by denitrification or photosynthesis. The host, in turn, may protect thesymbiont from competition, the environment, and predation. Despite rapid advances insampling, molecular, and microscopy methods, as well as the associated broadening oftheknown diversity of anaerobic ciliates, many aspects of these ciliate symbioses, including host specificity and coevolution, remain largely unexplored. Nevertheless, with the number ofcomparative genomic and transcriptomic analyses targeting anaerobic ciliates and their symbionts on the rise, insights into the nature of these symbioses and the evolution of the ciliate transition to obligate anaerobiosis continue to deepen. This review summarizes the current body of knowledge regarding the complex nature of symbioses in anaerobic ciliates, the diversity ofthese symbionts, their role in the evolution ofciliate anaerobiosis and their significance inecosystem-level processes.

P66 is a bacterial “don’t eat me signal” that mimics mammalian CD47 and facilitates immune evasion by Borrelia burgdorferi.

Abstract Innate immunity, the first line of defense against pathogens, relies on efficient elimination of the invading agents by phagocytes. Thus in the co-evolution of host and pathogen, pathogens developed mechanisms to dampen and evade phagocytic clearance. Here, we report that bacterial pathogens can evade clearance by macrophages through molecular mimicry of a mammalian anti-phagocytic “don’t eat me” signal. Using a high affinity structural probe for human CD47, a dominant “don’t eat me” signal, we discovered a bacterial protein that mimics CD47’s structure on the surface of Borrelia burgdorferi (Bb), a bacterial spirochete that can establish infection in mammals including Lyme Disease (LD). Blockade of the mimic promotes clearance of the infection in vivo. We identified P66, a known virulence factor, as the bacterial mimic of CD47. Finally, we determined that patients who return to health following LD infection are more likely to generate antibodies to p66 compared to patients who do not. This study demonstrates molecular mimicry as a means used by Bb to inhibit macrophages and evade phagocytic clearance; this mechanism may have broad implications for understanding host-pathogen interactions and the development of therapeutic strategies to combat bacterial infection. Research reported in this publication was supported by the Fairbairn family foundation; Bay Area Lyme Foundation; the Younger family foundation; the Robert J. Kleberg, Jr., and Helen C. Kleberg Foundation; the Virginia and D. K. Ludwig Fund for Cancer Research; AML grant R01CA086017; the PCBC from NIHLB U01HL099999; as well as grant U19AI109662. M.C.T. was supported by Stanford Immunology training grant 5T32AI007290, and the NIH NRSA 1 F32 AI124558-01 award. L.B.T.D. was supported by a Stanford Diversifying Academia Recruiting Excellence fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The Study of Microbe-Host Two-Way Communication.

Back-and-forth intercommunication in host–pathogen interactions has long been recognized to play an important role in commensalism and microbial pathogenesis. For centuries, we have studied these microbes in our surroundings, yet many questions about the evolutionary cross-talk between host and microbe remain unanswered. With the recent surge in research interest in the commensal microbiome, basic immunological questions have returned to the fore, such as, how are vast numbers of microbes capable of coexisting within animals and humans while also maintaining a healthy functional immune system? How is the evasion and subversion of the immune system achieved by some microbes but not others? The intricate and important-to-remember two-way interaction and coevolution of host and microbe is the communication network we must tap into as researchers to answer these questions.

Biodiversity of Mycobacterium tuberculosis in Bulgaria Related to Human Migrations or Ecological Adaptation.

Bulgaria is among the 18 high-priority countries of the WHO European Region with high rates of tuberculosis. The causative agent of tuberculosis is thought to have emerged in Africa 70,000 years ago, or during the Neolithic age, and colonized the world through human migrations. The established main lineages of tuberculosis correlate highly with geography. The goal of our study was to investigate the biodiversity of Mycobacterium tuberculosis in Bulgaria in association with human migration history during the last 10 centuries. We analyzed spoligotypes and MIRU-VNTR genotyping data of 655 drug-sensitive and 385 multidrug-resistant M. tuberculosis strains collected in Bulgaria from 2008 to 2018. We assigned the genotype of all isolates using SITVITWEB and MIRU-VNTRplus databases and software. We investigated the major well-documented historical events of immigration to Bulgaria that occurred during the last millennium. Genetic profiles demonstrated that, with the exceptions of 3 strains of Mycobacterium bovis and 18 strains of Lineage 2 (W/Beijing spoligotype), only Lineage 4 (Euro-American) was widely diffused in Bulgaria. Analysis of well-documented immigrations of Roma from the Indian subcontinent during the 10th to the 12th centuries, Turkic peoples from Central Asia in the medieval centuries, and more recently Armenians, Russians, and Africans in the 20th century influenced the biodiversity of M. tuberculosis in Bulgaria but only with genotypes of sublineages within the L4. We hypothesize that these sublineages were more virulent, or that ecological adaptation of imported M. tuberculosis genotypes was the main driver contributing to the current genetic biodiversity of M. tuberculosis in Bulgaria. We also hypothesize that some yet unknown local environmental factors may have been decisive in the success of imported genotypes. The ecological factors leading to local genetic biodiversity in M. tuberculosis are multifactorial and have not yet been fully clarified. The coevolution of long-lasting pathogen hosts should be studied, taking into account environmental and ecological changes.

Recombination Marks the Evolutionary Dynamics of a Recently Endogenized Retrovirus.

All vertebrate genomes have been colonized by retroviruses along their evolutionary trajectory. Although endogenous retroviruses (ERVs) can contribute important physiological functions to contemporary hosts, such benefits are attributed to long-term coevolution of ERV and host because germline infections are rare and expansion is slow, and because the host effectively silences them. The genomes of several outbred species including mule deer (Odocoileus hemionus) are currently being colonized by ERVs, which provides an opportunity to study ERV dynamics at a time when few are fixed. We previously established the locus-specific distribution of cervid ERV (CrERV) in populations of mule deer. In this study, we determine the molecular evolutionary processes acting on CrERV at each locus in the context of phylogenetic origin, genome location, and population prevalence. A mule deer genome was de novo assembled from short- and long-insert mate pair reads and CrERV sequence generated at each locus. We report that CrERV composition and diversity have recently measurably increased by horizontal acquisition of a new retrovirus lineage. This new lineage has further expanded CrERV burden and CrERV genomic diversity by activating and recombining with existing CrERV. Resulting interlineage recombinants then endogenize and subsequently expand. CrERV loci are significantly closer to genes than expected if integration were random and gene proximity might explain the recent expansion of one recombinant CrERV lineage. Thus, in mule deer, retroviral colonization is a dynamic period in the molecular evolution of CrERV that also provides a burst of genomic diversity to the host population.

Maximum antigen diversification in a lyme bacterial population and evolutionary strategies to overcome pathogen diversity.

Natural populations of pathogens and their hosts are engaged in an arms race in which the pathogens diversify to escape host immunity while the hosts evolve novel immunity. This co-evolutionary process poses a fundamental challenge to the development of broadly effective vaccines and diagnostics against a diversifying pathogen. Based on surveys of natural allele frequencies and experimental immunization of mice, we show high antigenic specificities of natural variants of the outer surface protein C (OspC), a dominant antigen of a Lyme Disease-causing bacterium (Borrelia burgdorferi). To overcome the challenge of OspC antigenic diversity to clinical development of preventive measures, we implemented a number of evolution-informed strategies to broaden OspC antigenic reactivity. In particular, the centroid algorithm—a genetic algorithm to generate sequences that minimize amino-acid differences with natural variants—generated synthetic OspC analogs with the greatest promise as diagnostic and vaccine candidates against diverse Lyme pathogen strains co-existing in the Northeast United States. Mechanistically, we propose a model of maximum antigen diversification (MAD) mediated by amino-acid variations distributed across the hypervariable regions on the OspC molecule. Under the MAD hypothesis, evolutionary centroids display broad cross-reactivity by occupying the central void in the antigenic space excavated by diversifying natural variants. In contrast to vaccine designs based on concatenated epitopes, the evolutionary algorithms generate analogs of natural antigens and are automated. The novel centroid algorithm and the evolutionary antigen designs based on consensus and ancestral sequences have broad implications for combating diversifying pathogens driven by pathogen–host co-evolution.

Pyricularia sp. jiangsuensis, a new cryptic rice panicle blast pathogen from rice fields in Jiangsu Province, China.

Pyricularia oryzae is a multi-host pathogen causing cereal disease, including the devastating rice blast. Panicle blast is a serious stage, leading to severe yield loss. Thirty-one isolates (average 4.1%) were collected from the rice panicle lesions at nine locations covering Jiangsu province from 2010 to 2017. These isolates were characterized as Pyricularia sp. jiangsuensis distinct from known Pyricularia species. The representative strain 18-2 can infect rice panicle, root and five kinds of grasses. Intriguingly, strain 18-2 can co-infect rice leaf with P. oryzae Guy11. The whole genome of P. sp. jiangsuensis 18-2 was sequenced. Nine effectors were distributed in translocation or inversion region, which may link to the rapid evolution of effectors. Twenty-one homologues of known blast-effectors were identified in strain 18-2, seven effectors including the homologues of SLP1, BAS2, BAS113, CDIP2/3, MoHEG16 and Avr-Pi54, were upregulated in the sample of inoculated panicle with strain 18-2 at 24 hpi compared with inoculation at 8hpi. Our results provide evidences that P. sp. jiangsuensis represents an addition to the mycobiota of blast disease. This study advances our understanding of the pathogenicity of P. sp. jiangsuensis to hosts, which sheds new light on the adaptability in the co-evolution of pathogen and host.

Orchid Root Associated Bacteria: Linchpins or Accessories?

Besides the plant-fungus symbiosis in arbuscular mycorrhizal (AM) and ectomycorrhizal (EM) plants, many endorhizal and rhizosphere bacteria (Root Associated Bacteria, or RAB) also enhance plant fitness, diversity, and coexistence among plants via bi- or tripartite interactions with plant hosts and mycorrhizal fungi. Assuming that bacterial associations are just as important for the obligate mycorrhizal plant family Orchidaceae, surprisingly little is known about the RAB associated with orchids. Herein, we first present the current, underwhelming state of RAB research including their interactions with fungi and the influence of holobionts on plant fitness. We then delineate the need for novel investigations specifically in orchid RAB ecology, and sketch out questions and hypotheses which, when addressed, will advance plant-microbial ecology. We specifically discuss the potential effects of beneficial RAB on orchids as: (1) Plant Growth Promoting Rhizobacteria (PGPR), (2) Mycorrhization Helper Bacteria (MHB), and (3) constituents of an orchid holobiont. We further posit that a hologenomic view should be considered as a framework for addressing co-evolution of the plant host, their obligate Orchid Mycorrhizal Fungi (OMF), and orchid RAB. We conclude by discussing implications of the suggested research for conservation of orchids, their microbial partners, and their collective habitats.