Plant associated protists—Untapped promising candidates for agrifood tools

Abstract The importance of host‐associated microorganisms and their biotic interactions for plant health and performance has been increasingly acknowledged. Protists, main predators and regulators of bacteria and fungi, are abundant and ubiquitous eukaryotes in terrestrial ecosystems. Protists are considered to benefit plant health and performance, but the community structure and functions of plant‐associated protists remain surprisingly underexplored. Harnessing plant‐associated protists and other microbes can potentially enhance plant health and productivity and sustain healthy food and agriculture systems. In this review, we summarize the knowledge of multifunctionality of protists and their interactions with other microbes in plant hosts, and propose a future framework to study plant‐associated protists and utilize protists as agrifood tools for benefiting agricultural production.


INTRODUCTION
Living plants are hosts of a complex microbiome, comprising of bacteria, fungi, archaea, protists and viruses that internally and externally colonize plant tissues (Hassani et al., 2018;Sapp et al., 2018;Trivedi et al., 2020). These beneficial, neutral and pathogenic plant-associated microorganisms can significantly influence plant health and performance. The plant hosts and their associated microbiomes are suggested to form a 'holobiont', where complex plant-microbe interactions play crucial roles in regulating and promoting plant growth, biogeochemical cycling, nutrient acquisition, fitness and protection, stress tolerance and disease suppression (Hassani et al., 2018;Liu et al., 2019). Plant associated microbiota, in some cases, even contribute more to plant protection and stress resistance than the defensive capacity of plant hosts (Hubbard et al., 2019). A holistic microbiome perspective to decipher the mechanisms that govern the assembly, interactions and functions of plantassociated microbiota, therefore, is a prerequisite to facilitate translational research and develop microbiome-based tools to enhance plant productivity and agricultural sustainability.
A panoramic view of the plant microbiota cannot be complete without considering protists as a pivotal component. Bacteria dominate the plant microbiota, followed by fungi, while protists and other organisms (e.g., archaea, nematodes and other soil invertebrates) are less abundant, but they were shown to be crucial in plant health and performance (Leach et al., 2017;Chen et al., 2021). Bacteria and fungi in the rhizosphere are enriched by carbon sources stemming from root exudates of plants (via bottom-up control), however, they are major microbial prey for protists and thus subject to top-down control by protist consumers. Protists, representing the vast diversity of unicellular eukaryotes, function as consumers (main predators of bacteria, fungi and small animals), primary producers (important carbon fixers via photosynthesis), plant and animal parasites, and decomposers (Geisen et al., 2018). The contributions of protists to nutrient input, organic matter decomposition and plant health have been previously reported (Bonkowski, 2004;Xiong et al., 2020;Geisen et al., 2021). Nonetheless, plant-associated protists and their functions for plant hosts, compared to bacteria and fungi, have been largely underestimated (Gao et al., 2019;Trivedi et al., 2020). While the importance of plant beneficial microorganisms as promising agrifood tools to improve crop production and agricultural sustainability has been increasingly recognized Hu et al., 2022), the plant-protist-microbe interactions in the above-and below-ground systems are not well understood.

POTENTIAL FUNCTIONS AND INTERACTIONS OF PROTISTS IN THE PLANT-SOIL-MICROBE NETWORK
Although protists have the great potential to improve nutrition, suppress pathogens, promote plant growth, and function as bioindicators for plant health (Bonkowski, 2004;Xiong et al., 2020), protists colonizing inside plant tissues remain vastly untapped. Most studies on plant-associated protists have focused on plant pathogenic or parasitic protists causing plant diseases (Dumack & Bonkowski, 2021) or the belowground protist community particularly in the rhizosphere (Fiore-Donno et al., 2022). Bacteria and fungi are wellcharacterized plant microbiome components with distinct community compositions across different compartments (e.g., phyllosphere, anthosphere, leaf and root endospheres, rhizosphere, and bulk soils) (Liu et al., 2019;Trivedi et al., 2020;Sun et al., 2021b). Given the selective feeding preference of different protist groups for bacteria and fungi (Dumack et al., 2020), plant compartments at different developmental stages may harbour distinct taxonomic and functional diversity, community structure and functions of protists. In this article, we discuss known functions of protists and propose their potential roles and activities in different compartments of plants ( Figure 1; Table 1).

Phyllosphere-associated protists
Protists form key members of the plant microbiome and an external force shaping the plant microbiome assembly (Geisen et al., 2018;Gao et al., 2019), but the diversity and feedback of protists on phyllosphere microbiome remain surprisingly unknown. The occurrence of a protist strain Colpoda cucullus in leaves and stems in the 1970 s is one of early findings about the phyllosphere-associated protists (Bamforth, 1973). Protists, especially the phylum Cecozoa consumers, have been recently identified in the model plant Arabidopsis thaliana (Sapp et al., 2018), sorghum (Sun et al., 2021a), grasses, legumes and forbs (Flues et al., 2017;Flues et al., 2018), with the ability to improve plant growth and biomass. The phyllosphere, a habitat of various phages, prokaryotes, protists, fungi, and visiting insects (e.g., bees, butterflies and herbivores), is supposed to regulated by their complex trophic interactions under direct impacts of environmental changes. Protists shape the community composition and activities of bacteria and fungi through selective predation (Bonkowski, 2004;Gao et al., 2019). Notably, the selective predation of protists triggers distinct bacterial strains to produce antimicrobials, such as 2,4-diacetylphloroglucinol (DAPG) and pyrrolnitrin (Jousset et al., 2006), or violacein (Matz et al., 2004), which has been recorded in the interaction between one or a few model protist and bacterial species under in vitro conditions. Hence, protists may stimulate bacteria or fungi to excrete toxic metabolites to protect plants from air-borne pathogens or herbivores.
Furthermore, protists can potentially select beneficial traits of microbes through (i) promoting phytohormoneproducing bacteria and ultimately enhancing plant fitness and development; and (ii) regulating the metabolic and functional profiles of bacterial community in the phyllosphere ( Figure 1). Some first evidence about phytohormone stimulation of protists has been found in plant rhizosphere, and their beneficial effects on plant hormones in the phyllosphere are a fertile area to discover. Recent studies have indicated that bacterivorous amoebae promoted bacteria producing essential phytohormones (auxin and cytokinin) in the plant rhizosphere though protists alone cannot produce plant hormones (Bonkowski & Brandt, 2002;Krome et al., 2010). Flues et al. (2017) revealed that, through a shotgun metagenomic sequencing, the predation of leaf-associated protists Cercomonas and Paracercomonas strains (Cercozoa) dramatically influenced the taxonomic composition and metabolic functions of leaf-associated bacterial community under in vitro conditions, suggesting the strong regulation of protists on the activities and functions of bacteria in the phyllosphere. Many other representatives of leaf-associated Cercozoan consumers (Rhogostoma spp.) were found to feed on fungi (here are yeasts) and algae in the phyllosphere of A. thaliana, and this grazing activity indicated crucial effects of protists on a wide range of microbes in the phyllosphere.
Plants are not passively benefited by microorganisms but may proactively use the strategy 'cry for help' to recruit beneficial microorganisms to protect themselves under the abiotic (e.g., drought or high temperature) and biotic stresses (e.g., pathogens or herbivores). The underlying mechanisms and recruited microorganisms of this strategy, however, are unclear and probably distinct across plant compartments. Strikingly, a board spectrum of bacteria and fungi (e.g., yeasts) inhabit the anthosphere (i.e., flowers and surrounding zones), especially nectar, pollen (Vannette et al., 2013;Schaeffer et al., 2017) and flower surface (Ushio et al., 2015;Arunkumar et al., 2019), which significantly influence flower-pollinator interactions, plant reproduction and yield. Due to the diverse microbes transmitted from various sources, flowers are potentially dynamic hubs of microbes and pollinators. However, the diversity and roles of protists in the anthosphere are far from being fully elucidated.
Moreover, endophytic protists colonize root and leaf and stem endosphere, where their interplay with plant hosts and other microbes can possibly influence plant hormones, defensive systems and nutrient translocation to every plant tissue. The stimulation of uptake and translocation of nitrogen from rhizosphere soils, plant roots to shoots by protists were reported in wheat plants (Clarholm, 1985;Henkes et al., 2018). Notably, the amoebae Acanthamoeba castellanii promoted the phytohormone production (auxins and cytokinin) of bacteria in the phyllosphere of cress (Lepidium sativum L.) and A. thaliana (Krome et al., 2010). Most recent studies have attempted to characterize the compositions of F I G U R E 1 Functions and interactions of protists within the plant-associated microbiota in different plant compartments (phylloshere, anthosphere, leaf, stem and root endosphere, rhizosphere and bulk soil). (i) Through predation or symbiosis, protists interact with bacteria, fungi (especially, arbuscular mycorrhiza fungi (AMF)) and other microbes (e.g., archaea) in cycling, uptake and/or translocation of essential nutrients (e.g., nitrogen, phosphorous, carbon, silicon, calcium, magnesium and iron) for plants and soil organisms in rhizosphere and bulk soils. Protists can also enhance nutrient input as carbon fixers or through releasing nutrients after the prey consumption; (ii) protists have the potential to form symbiotic or facilitative relationships with nitrifying and ammonifying bacteria or archaea in nitrogen fixation in plant rhizosphere; (iii) the predation of protists can trigger the antimicrobial production of bacteria or fungi, inhibiting the infection of air-borne or soil-borne pathogens or pests in phyllosphere and rhizosphere; (iv) protists may also enhance plant hormone and stress tolerance by directly interacting with plant hosts or stimulating plant-beneficial traits of microbes.
protists in the plant microbiome (Dumack et al., 2022;Sun et al., 2021a), hence further insights into the multitrophic interactions of protists with plants, microbes, airborne pathogens and insects in the phyllosphere are required.

Rhizosphere-associated protists
In contrast to other plant compartments, protists in the rhizosphere have received more attention with growing evidence for their crucial roles in (i) plant health and  (Bonkowski et al., 2000) Spruce seedlings Rhizosphere (root systems); phyllosphere (stems and needles) Acanthamoeba sp. Plant phosphorus and calcium uptake and translocation; Biomass of plant compartments (Bonkowski et al., 2001) Rice plants (Oryza sativa L.)

Rhizosphere (plant roots) Acanthamoeba castellanii
Plant hormone production (auxin, indolyl-3-acetic acid (IAA)); Root growth and architecture (Bonkowski & Brandt, 2002) disease control (Xiong et al., 2020), (ii) nutrient cycling (Clarholm, 1985;Bonkowski, 2004), and (iii) plant hormones and growth (Bonkowski & Brandt, 2002). Many bacterial and fungal taxa are well-known producers of antibiotics and toxic metabolites (Hutchings et al., 2019). The selective predation or even the presence of protists can trigger bacteria to produce specific antibiotics as weapons to kill or avoid protists through species-specific response (Nguyen et al., 2020). For instance, Pseudomonas fluorescens strain SS101 and Pseudomonas fluorescens strain SBW25 produced antibiotics massetolide and viscosin, respectively, in response to the same bacterivorous amoeba Naegleria americana C1 (Mazzola et al., 2009;Song et al., 2015). Fungi also emit antimicrobial volatiles to inhibit the bacterial motility or growth upon bacterial-fungal interaction (Rybakova et al., 2017;Bruisson et al., 2020). However, there is a paucity of effects of protists on the antibiotic excretion of fungi. The antibiotics produced by bacteria and fungi are considered as a defensive mechanism to toxify not only protists but also other microbial competitors in natural habitats (Święciło, 2016;Cruz-Loya et al., 2019). Through this effect, when plants 'cry for help' by sending signals via root exudates (volatiles, organic acids or others) under pathogen or pest attacks (Liu et al., 2019), protists may respond by recruiting antibiotic producers to produce antimicrobials to inhibit pathogens or pests for plant protection. However, this strategy of plants and their associations with protists are still elusive questions.
As primary microbial predators, protists can also directly consume bacterial and fungal pathogens. The consumptive effect of protists, typically protistan consumers, can cause fatality of a wide range of bacterial and fungal strains (Chakraborty et al., 1983;Dumack et al., 2016). In the rhizosphere of A. thaliana, the diversity and abundance of specific bacteria taxa, especially Betaproteobacteria and Firmicutes, were significantly decreased under the predation of soil amoeba A. castellanii. Bahroun et al. (2021) reported that bacterivorous protists alone and their synergistic interactions with bacteria reduced disease severity caused by a fungal pathogen Fusarium solani S55 and improved root length and plant growth of faba bean (Vicia faba) seedlings (Table 1). Recent studies have indicated important links of protists to soil-borne disease control and plant health in the rhizosphere of tomatoes (Xiong et al., 2020), cucumber  and banana plants (Guo et al., 2022). In particular, numerous Cercozoan and Amoebozoan species can function as important indicators for the health of tomato plants. Guo et al. (2022) also revealed that the protistan consumer Cercomonas lenta strain ECO-P-01 substantially suppressed the density of the fungal pathogen Fusarium oxysporum and increased the disease-suppressive bacteria Bacillus in the rhizosphere, and subsequently improved banana plant growth and yield. Hence, a comprehensive understanding of protists in the rhizosphere and other plant compartments will promote their applications in plant disease suppression.
Protists are also pivotal contributors to nutrient cycling in the rhizosphere (Table 1). Nutrients are temporarily locked up in rhizosphere bacterial and fungal biomass and can be translocated to protists as microbial feeders or unlocked by the protists' predation and eventually channelled to benefit plants, which is called 'the microbial loop' (Clarholm, 1985). Protists directly release nitrogen and carbon after prey digestion or form a symbiotic relationship with beneficial fungal or bacterial taxa in cycling essential nutrients (nitrogen, carbon, iron, silicon or phosphorous) (Geisen et al., 2018;Gao et al., 2019), enhancing soil nutrient input and fertility for nurturing plant growth and rhizo-microbiome. The great contribution of protists to nutrient cycling has long been recognized since 1985, when Clarholm demonstrated the increasing nitrogen uptake to 75% by plants under the inoculation of protists. The presence of protists promoted plant phosphorus and calcium uptake and translocation to stems or needles, as well as modulated nutrient concentrations (nitrogen, phosphorus, carbon to nitrogen ratio (C/N ratio), calcium and magnesium) (Bonkowski et al., 2001). Consequently, this regulation of protists led to the improvement of root growth and architecture as well as biomass of different compartments (shoots, roots and needles) of spruce seedlings. A similar beneficial effect of protists was found in rice plants (Oryza sativa L.) (Henkes et al., 2018). Moreover, phototrophic protists contribute to carbon cycling as carbon fixers via photosynthesis (Schmidt et al., 2016), providing nonnegligible carbon and oxygen inputs to rhizosphere organisms and the basis for soil life, but their capacity for carbon sequestration is still unknown.
Notably, benefits of protists to plant nutrition are more efficient when forming symbiosis with other microbes, particularly arbuscular mycorrhizal fungi (AMF) that enhance plant nitrogen and phosphorus uptake. Protists might facilitate nutrient acquisition, mineralization and translocation of AMF (Zuccaro et al., 2014;Henkes et al., 2018), and promote the growth and activities of nitrifying bacteria and other bacteria (Bonkowski, 2004), suggesting intimate protist-microbe links in plant benefits. For instance, Bonkowski et al. (2001) indicated that the joint effects of protists and mycorrhiza significantly enhanced the phosphorous uptake from roots to stems, as well as affected rhizosphere microbes and essential plant nutrients (carbon, phosphorous and trace elements), which maximized the biomass of different spruce compartments (shoots, stems and needles). Protists, in rumen ecosystems, were detected to have positive links to archaea (Solomon et al., 2022), which are key players in the global nitrogen cycle (Hu et al., 2015). However, the contribution of archaea to plant hosts and protist-archaea relationships in nutrient cycle is an intriguing unexplored topic. Upon nutrient shortage, beneficial protist-microbe interactions may be boosted by the plant strategy 'cry for help', and we cannot have a full understanding of protists' roles if ignoring their contributions.
Protists can also significantly influence plant hormones and development through regulating the community structure and activities of plant-hormone producing rhizobacteria. Plant growth-promoting phytohormones auxins (indolyl-3-acetic acid (IAA)) were found in the inoculation of the most studied model species A. castellanii in bacterial cultures (Nikoljuk, 1969) and rhizosphere of watercress seedlings (L. sativum) by modulating phytohormone-producing bacteria or rhizobacterial community (Bonkowski & Brandt, 2002). While root systems are paramount apparatus to take up and allocate water and nutrients to every plant tissue for plant growth and environmental adaptation, protists, such as A. castellanii, can trigger phytohormone production (auxins and cytokinin) of bacteria, resulting in the enhancement of root growth and architecture and development of plants L. sativum and A. thaliana, more than bacteria standalone (Bonkowski & Brandt, 2002;Krome et al., 2010). Interestingly, the regulation on phytohormone-producing bacteria strengthens root growth and architecture of many crops, including watercress, pea and cress (Table 1). Hence, it is evident that soil-or rhizosphere-associated protists can significantly influence both the above-and below-ground compartments of the plant hosts. While microbes are acknowledged as important hormone producers of plants (Nakano et al., 2022), more explorations of protists' roles in the inter-organismal phytohormone networks between plant hosts, protists and other microbes are critical to deploy beneficial protists in improving plant immunity and development.

Protists in bulk soils
In bulk soils, the diversity of protists is higher than that in the rhizosphere, root and litter (Ceja-Navarro et al., 2021;Fiore-Donno et al., 2022), which indicates that soil protists function as a 'microbial seed bank' for plant support and soil functions, as well as the selection of plants for protist communities. Moreover, protists can influence elemental cycles, soil fertility and soil microbiome by (i) steering the composition and activities of beneficial microorganisms (e.g., AMF or nitrifying microbes), (ii) excreting nitrogen or carbon sources after the predation and consumption of prey in bulk soils, and (iii) mediating the community composition and interactions of soil microbiome via facilitative, symbiotic or predatory relationships between protists and other microbes. The positive relationships between protists and bacteria have been identified in soil ecosystems (Nguyen et al., 2021), but further research is required to disentangle mechanisms for the interplay and roles of protists, fungi, bacteria, archaea, and viruses in plant-associated microbiota. Beside the aforementioned benefits, parasitic protists have negative effects on plants, as pathogens have been more thoroughly characterized than neutral and beneficial protists (Dumack & Bonkowski, 2021). A large number of non-pathogenic endophytic protists inhabit plant tissues and across rainforest soil ecosystems (Mahé et al., 2017), but their identity and functions on plant hosts remain unknown. Given their high abundance in natural habitats, we suppose that endophytic protists have unexplored benefits to plant hosts.

FUTURE FRAMEWORK FOR UNRAVELLING THE ROLES OF PLANT-ASSOCIATED PROTISTS IN PLANT HEALTH AND PRODUCTION IMPROVEMENT
Protists alone or their interactions with other microbes are considered to play crucial roles in the plant holobiont. It is promising to develop protist-based tools to enhance nutrient availability and plant growth as biofertilizers, to control plant disease infection and microbial functions as biocontrol agents, or to promote plant hormones and nutrient cycling activities and survival of plant beneficial microbes in modern agriculture. Compared to other plant microorganisms, the functions, signalling and feedbacks of protists in multi-organismal (host-protist, protist-microbe, and protist-visiting insects) interactions with or without the infection of soilborne or air-borne plant pathogens and pests are largely unexplored. A more comprehensive understanding of the molecular mechanisms and functions of plant-protist-microbe interactions will enable us to steer the activities and performance of microbes in the plant holobiont. Therefore, we propose and discuss future frameworks to generate a holistic view of plantassociated protists and the manipulation and applications of protist-based models in crop production, namely: (i) identification of key factors structuring the taxonomic and functional traits of plant-associated protists as well as the core and keystone taxa of protists; (ii) isolation and selection of plant beneficial protists for various crops under different stresses; and (iii) establishment and applications of protist-based synthetic communities (SynComs) to improve plant performance ( Figure 2).
Firstly, the identification of key factors structuring the taxonomic and functional traits of plant-associated protists is a crucial step. To date, most studies have characterized plant-associated protists by conventional (microscopy-based and direct counting) methods, quantitative PCR or amplicon sequencing. Identifying microbial eukaryotes with high throughput sequencing techniques, however, is not straight forward, since severe primer-biases were identified in previous protist surveys (Lentendu et al., 2014;Hirakata et al., 2019). For instance, although many soils are known to be dominated by protists of the taxa Amoebozoa and Cercozoa, the primer-based surveys constantly underestimate the importance of Amoebozoa (Bonkowski et al., 2019). Metatranscriptomics can overcome this issue as they do not rely on primers and, in accordance to what is found by morphological surveys, Amoebozoa may dominate in such datasets (Urich et al., 2008;Geisen et al., 2015). Furthermore, it is still difficult to estimate exact functioning of protists. Trait databases are helpful for the exploration of functioning in microbial eukaryotes (Dumack et al., 2020), but there is still a lack of a database covering all distinct protistan taxa. The characterization of protists in different plant compartments (including phyllosphere, leaf, stem and root endosphere, rhizosphere soil and bulk soil) in largescale field investigations is important to have full understandings about their taxonomic and functional diversity F I G U R E 2 Proposed framework for future studies of plant-associated protists and harnessing protist-based products for improving crop production: (1) identification of key factors structuring the taxonomic and functional traits of plant-associated protists and plant core protists in large-scale investigations; (2) isolation and selection of potential plant beneficial protist candidates through data integration and preliminarily tests in short-term controlled conditions; (3) establishment and applications of protist-based synthetic communities (SynComs) to improve plant performance in monoculture or crop mixture. Different SynComs constituted by different inoculants: single protist species, different protist species, protists-bacteria, protists-bacteria-fungi, and protists-fungi. The integration of 'omics' techniques (i.e., metatranscriptomics, metaproteomics and metabolomics) with integrated data analysis characterizes cellular activities, functions and metabolites of protists, plant host and other organisms in the plant holobiont. The selection of crop species in this figure is just for illustration. and community compositions for each plant species. The combination with co-occurrence networks and statistical modellings will further disentangle the key drivers and principles shaping the protist community assembly and dynamics in plant microbiome, as well as build up a database of key protists that best predict plant performance parameters.
Many persistent and abundant members of a specific host found across wide-range habitats constitute a core plant microbiota, which carry essential genes to support plant fitness as well as play crucial roles in maintaining multiple functions and stability of the host microbiome (Shade & Stopnisek, 2019). Core bacterial taxa, for example, members of the orders Rhizobiales and Pseudomonadales, are reported to benefit plant fitness, growth and resilience under stresses (Trivedi et al., 2020). Notably, keystone taxa of protists, highly associated members regardless of their abundance, deserve special attention because they crucially affect community structure and functions (Banerjee et al., 2018). Therefore, the determination of core and keystone taxa of protists for major crops across different regions, along with core and keystone taxa of bacteria and fungi (Banerjee et al., 2018;Trivedi et al., 2020), will leverage our capacity to manipulate plant microbial activities and design optimal SynCom models for maximizing growth and yields of specific crops. Crucial for this is a coupling of high throughput sequencing and metatranscriptomic approaches with subsequent culture attempts, first to identify core symbionts and then to provide them as a culture to research.
Secondly, to incorporate protists into the agrifood toolbox, it is paramount to establish a collection of plant beneficial protists for various crops under different stress conditions. It is promising to tailor the highthroughput isolation approach which has proved to be effective in isolating bacterial strains from root microbiota (Zhang et al., 2021), to characterize and isolate protists from various plant tissues (e.g., leaf and stem endophytes). In the first selection step, the data integration of plant protists from large-scale investigations with findings of the high-throughput protist isolation will be a crucial reference for selection and nomination of promising protist species to establish protist-based Syn-Coms for improving plant performance. In the second selection step, the selected protist species, alone or in a subset of core and keystone protists, can be preliminarily tested for their capacity in performing desired functions, such as suppression of common fungal pathogens and resistance to abiotic stresses, in short-term controlled laboratory conditions. Core and keystone taxa of protists conferring desired plant-beneficial functions will be considered as key members of the protistbased SynComs. However, other core and keystone taxa of protists, which do not have the desired features in the preliminary tests, should not be discarded because their performance may be boosted in facultative or antagonistic interactions with specific microbes.
Thirdly, protist isolates alone or combined with beneficial bacterial or fungal strains are used to construct different protist-based synthetic communities to improve plant health and performance. These SynComs can mimic biological interactions (e.g., competition, predation or symbiosis) in natural settings, and the diversification of trophic interactions (e.g., bottom-up and top-down controls or trophic cascade) will boost microbes to produce crucial products (e.g., phytohormones, antibiotics and other compounds), and consequently stabilize the phytobiome and promote crop development. We propose to apply the protist-based SynCom models for monoculture or mixture of plant species. Neighbouring crops in the plant mixture can increase interspecific interactions and functions of beneficial microbes, and plant uptake of essential resources (nutrients or water), with positive consequences for disease suppression and plant growth (Jing et al., 2022). All SynComs will be assessed for their efficacy in benefiting plant fitness and growth, nutrient cycling and uptake, disease controls and stress tolerance for each plant species. Some protist-based products, for example, have hit the market and been applied in crop production, such as a protist species Nosema Iocustae as a biological control agent in over 90 species of grasshoppers, locusts, and crickets in the United States (https://www.gardeninsects.com/ grasshopperbait.asp); and 19 biofertilizers developed from a mixture of beneficial protists, bacteria and fungi enhancing nutrients, plant growth and resilience for a variety of crops in Netherlands (https://ecostyle.nl/ zoeken?query=protozoa).
In this step, the integration of multiple 'omics' techniques (including metatranscriptomics, metaproteomics and metabolomics) with machine learning and statistical modelling, rather than amplicon sequencing or one single method, will enable us to characterize the panoramic profile of cellular activities, functions, molecular signalling and metabolites of protists, plant host and other organisms in the plant holobiont. The metatranscriptomics elucidate the microbial identification, gene expression and functional profile of protists and other organisms, while metaproteomics (e.g., matrix-assisted laser desorption-ionization time of flight (TOF)/TOFmass spectrometry (MS)) is powerful to unravel protein identification, quantification and origin (Wang et al., 2011). Metabolomics can detect and quantify untargeted primary metabolites (e.g., organic acids, amino acids, and others), such as by gas chromatography (GC)-MS, and secondary metabolites produced by plant hosts, protists and other associated microorganisms, such as by liquid chromatography (LC)-highresolution MS (LC-HRMS) (Weckwerth, 2010;Sumner et al., 2015). The application of machine learning and statistical modelling with transcriptomic, proteomic and metabolomic data can maximize our capacity to identify and predict compound composition, metabolic pathway, functional traits and activities of protists with the plant hosts and other organisms. For instance, the software METABOLIC is an advanced toolkit to profile metabolic and biogeochemical traits, and functional networks in microbial communities (Zhou et al., 2022). This integrated strategy will help us to explore and confirm the multifunctionality and benefits of plantassociated protists to plant hosts and understand the complex trophic interactions within the plant-soil system in a holistic manner.
The development of protist-based SynCom models as agrifood tools is potential to improve agricultural production. It is obvious that there will not be 'one size fits all' SynComs (Vorholt et al., 2017), hence the construction of protist-based solutions should target species-or tissue-specific SynComs for distinct plants at different developmental stages to optimize the efficacy in plant productivity, like commercial fertilizers or pesticides. Given the above-mentioned contributions of protists to plants, we advocate for future efforts to target the development of beneficial protists as novel and sustainable biofertilizers for improving plant growth and productivity, biological control agents for enhancing pathogenic defence, and biological stimulation strategies for boosting microbial activities and plantpromoting traits for plant health and performance. Biofertilizers are gaining interests across the agricultural sector, due to the recent rapid increase of fossil fuel price and fertilizer costs, the inoculation and formulation of protists into biofertilizers will be powerful to unlock natural nutrient sources or inorganic and organic fertilizers in soils. Nevertheless, the lack of sufficient knowledge about the roles of protists in soil ecology limits our ability to manage soil health for sustaining crop production. Future research on unravelling the functions and strategies of beneficial plant-associated protists is necessary to enhance plant health and production, thus reducing the application of fungicide and pesticides.

CONCLUDING REMARKS
Protists are key members of the plant-associated microbiota. It is evident that their contributions alone or in combination with other microorganisms significantly benefit plants in not only a single but multiple aspects, such as plant nutrition, disease control, plant health and performance. The interplay between the hosts and associated protists in different plant and soil compartments is complex and still far from being fully elucidated. Therefore, there are calls to disentangle driving factors and key roles of protists in ecological processes and agricultural productivity, which can provide new insights into the manipulation and applications of beneficial protists as biofertilizers and other agricultural products in benefiting crop health and productivity and ultimately sustaining healthy agricultural systems. Although the protists' benefits to plants as bio-fertilizers or biocontrol agents have been aware, we still have limitations on approaches studying the identity and functions of protists, as well as challenges in how to engineer efficient protist-based SynComs and how to maintain their persistence and efficacy in crop production. The innovation of plant-beneficial products from protists is a daunting task, but it will pay the ways to accelerate the development of protist-based products and to innovate novel mobile molecular technologies to quickly assess and monitor the activities and community composition of the applied beneficial microbiome in smart-farming systems and agricultural fields in the near future.
We also highlight some important questions about plant-associated protists in the plant holobiont: (1) What are biochemical or molecular signals that protists recruit or pay partnerships with other plant-associated microorganisms (e.g., bacteria or fungi) in benefiting plant hosts, as well as interact with insects (e.g., pollinators or ants) or herbivores? (2) What individuals or groups of protists are recruited by the plant hosts? (3) How do protists at different plant compartments respond to the strategy 'cry for help' of plants under biotic (pathogen or pest infection) or abiotic (e.g., low/high temperature, drought or salinity) stresses? (4) What are interactions between plant hosts and plant or soil microbiome under impacts of climate change? (5) Beside plant roots, do other plant tissues (e.g., leaf or stem) use a similar strategy 'cry for help' to interact with or recruit beneficial protists and other microorganisms for dealing with different stresses? (6) How can SynComs and other protist-based tools be safely introduced and applied to recipient soils and crops? (7) How can we estimate and maintain the efficiency and persistence of protist-based SynComs and other tools in enhancing plant growth and productivity in recipient soils and crops? The answer to these questions is a challenge but also a great opportunity to leverage our capacity to deploy plant-associated microbiota to improve crop health and performance. No single method but the integrated advanced approaches can help us fully understand complex interactions in the plant holobiont.