# Phytophthora zoospores: From perception of environmental signals to inoculum formation on the host-root surface

Ilaria Bassani, Marie Larousse, Quang Tran, Agnès Attard, Eric Galiana

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Ilaria Bassani, Marie Larousse, Quang Tran, Agnès Attard, Eric Galiana. Phytophthora zoospores: From perception of environmental signals to inoculum formation on the host-root surface. Computational and Structural Biotechnology Journal, 2020, 18, pp.3766-3773. 10.1016/j.csbj.2020.10.045. hal-03098346

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# Phytophthora zoospores: From perception of environmental signals to inoculum formation on the host-root surface

Ilaria Bassani<sup>a</sup>, Marie Larousse<sup>a</sup>, Quang D. Tran<sup>b,1</sup>, Agnès Attard<sup>a</sup>, Eric Galiana<sup>a,*</sup>

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## ARTICLE INFO

Article history:

Received 28 August 2020

Received in revised form 29 October 2020

Accepted 31 October 2020

Available online 21 November 2020

Keywords:

Phytophthora zoospore

Motion

Perception

Soil

Microbiota

Host-root

Taxis

## ABSTRACT

To explore moist soils and to target host plants, phytopathogenic *Phytophthora* species utilize the sensory and propulsion capabilities of the biflagellate unicellular zoospores they produce. Zoospore motion and interactions with the microenvironment are of primary importance for *Phytophthora* physiology. These are also of critical significance for plant pathology in early infection sequential events and their regulation: the directed zoospore migration toward the host, the local aggregation and adhesion at the host penetration site. In the soil, these early events preceding the root colonization are orchestrated by guidance factors, released from the soil particles in water films, or emitted within microbiota and by host plants. This signaling network is perceived by zoospores and results in coordinated behavior and preferential localization in the rhizosphere. Recent computational and structural studies suggest that rhizospheric ion and plant metabolite sensing is a key determinant in driving zoospore motion, orientation and aggregation. To reach their target, zoospores respond to various molecular, chemical and electrical stimuli. However, it is not yet clear how these signals are generated in local soil niches and which gene functions govern the sensing and subsequent responses of zoospores. Here we review studies on the soil, microbial and host-plant factors that drive zoospore motion, as well as the adaptations governing zoospore behavior. We propose several research directions that could be explored to characterize the role of zoospore microbial ecology in disease.

© 2020 The Author(s). Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Contents

<table><tr><td>1. Introduction</td><td>3767</td></tr><tr><td>2. The soil environment</td><td>3767</td></tr><tr><td>2.1. Impact of soil on zoospores</td><td>3767</td></tr><tr><td>2.2. Impact of zoospores on soil</td><td>3769</td></tr><tr><td>3. The microbial environment</td><td>3770</td></tr><tr><td>3.1. Zoospore-zoospore interactions</td><td>3770</td></tr><tr><td>3.2. Zoospore interactions with other microorganisms</td><td>3770</td></tr><tr><td>4. The plant environment: The rhizosphere</td><td>3771</td></tr><tr><td>5. Concluding remarks</td><td>3771</td></tr><tr><td>CREdiT authorship contribution statement</td><td>3772</td></tr><tr><td>Declaration of Competing Interest</td><td>3772</td></tr><tr><td>Acknowledgements</td><td>3772</td></tr><tr><td>References</td><td>3772</td></tr></table>

<footnote><sup>a</sup>Université Côte d'Azur, INRAE, CNRS, ISA, Sophia Antipolis 06903, France</footnote>

<footnote><sup>b</sup>Université Côte d'Azur, CNRS, UMR 7010, Institut de Physique de Nice, Nice 06108, France</footnote>

<footnote>* Corresponding author at: Institute Sophia Agrobiotech, INRAE PACA, 400 Route des Chappes, 06903 Sophia, Antipolis, France.</footnote>

<footnote>E-mail addresses: ilaria.bassani@inrae.fr (I. Bassani), marie.larousse@inrae.fr (M. Larousse), duc-quang.tran@pasteur.fr (Q.D. Tran), agnes.attard@inrae.fr (A. Attard), eric.galiana@inrae.fr (E. Galiana).</footnote>

<footnote n="1">¹ Present address: Institut Pasteur, CNRS UMR 3691, Cell Polarity, Migration and Cancer Unit, 75015 Paris, France.</footnote>
# 1. Introduction

Oomycetes of the genus *Phytophthora* comprise several of the most harmful plant pathogens described to date. They are responsible for serious diseases in hundreds of plant species, with massive ecological and economic losses worldwide [1,2]. Around 120 *Phytophthora* species have been described thus far [3]. Many environmental factors have been shown to affect *Phytophthora* disease development, including climatic, chemical, physical and biological conditions that can interact with one another to induce the onset of disease [4]. At the landscape scale, moisture and wind air speed, geomorphologic and topographic features, soil clay content, and the movement of animals and humans are all traits associated with *Phytophthora* epidemiology [5-7]. This review focuses on root diseases caused by *Phytophthora* zoospores and addresses recent findings on environmental signals that lead to inoculum formation on the host surface. The emergence of disease is controlled by close proximity between roots and water flows, allowing root-to-root contact and increasing the concentration or dispersal of propagules, in addition to plant-pathogen interaction [5]. At the microenvironmental scale, the disease risk starts as soon as zoospores escape from a sporangium. Indeed, while *Phytophthora* species grow as filamentous coenocytic hyphae and produce both sexual (oospores) and asexual (sporangia, zoospores) propagules, the epidemic spread of root diseases is mainly based on dispersal in soil and water films as biflagellate zoospores [1,8].

Zoospores are ellipsoidal, single nucleated cells that lack a cell wall. Each zoospore swims and explores randomly the environment by means of two flagella, one directing forward and the other one backward. Both flagella are inserted in a ventral groove [8] and are able to propel the cell body at high speed, up to 250 µm/s [9] (Fig. 1, Video S1 of Supplementary Data). For beating orchestration, the two flagella exhibit the same structure and repertoire of motor proteins as other eukaryotic microswimmers (such as *Chlamydomonas reinhardtii*), e.g. dyneins, which bind tubules under the control of radial spokes [1,8,10,11]. The specific opposing orientation and direction of beating patterns of the flagella make *Phytophthora* zoospores a simple but attractive model to investigate the hydrodynamics of microswimmers as they explore and invade a porous medium such as the soil. When beating, the two flagella orientate the wave propagation outwards from the cell body, giving the appearance that they are competing with each other. However, the smooth, whiplash-like posterior flagellum pushes water outwards in its wave propagation, while the anterior draws the fluid toward the body thanks to multiple mastigonemes attached along the flagellum (Fig. 1) [12]. Theoretical and biological studies have established the effect of mastigonemes in reversing the thrust generated by the anterior flagellum [12,13], with both flagella being found to generate thrust in the same direction following the wave propagation direction of the anterior flagellum. Thus, the actions of flagellar mastigonemes are critical in the determination of zoospore swimming direction, speed and propulsive efficiency.

Our basic molecular understanding of the perception of environmental signals by zoospores has been mainly generated by *in vitro* investigations that mimic natural conditions. In the rhizosphere, the first step toward a successful infection relies on the perception of diverse stimuli at multiple levels (Fig. 2). The ion exchange dynamics between soil particles and plant roots, together with the chemical gradients generated by root exudates, dictate the direction of motion (Fig. 2B,C) and activate cell responses. This results in coordinated zoospore behavior and their preferential localization to the water film at the interface between soil particles and plant roots [8,14,15]. The early stages of host surface colonization involve sequentially the loss of the two flagella, the discharging of adhesive molecules and the transition to walled cysts which undergo germination before penetration and colonization [1,8]. They may also involve zoospore population dynamics, where zoospores produce signals to attract hundreds of individuals, resulting in encystment, extracellular mucilage elaboration and biofilm formation on the plant surface [16]. Moreover, in soil, zoospores can either compete or cooperate with other rhizospheric microbiota species at the root surface (Fig. 2D) [17]. These interactions result in changes in microbiota composition [18], regulation of disease onset, as well as an additional and complex array of environmental signals that can both dictate motion direction and regulate the early steps of root surface colonization.

Soil-plant-zoospore-microbiota interactions are thus emerging as key events for *Phytophthora* dissemination, inoculum constitution and infection establishment. Here we review studies on environmental, microbial and host-plant factors that have been shown to drive the pre-infection behavior of zoospores before disease development. They mainly relate to (1) the displacement in the water film at the interface with soil particles; (2) the interactions with other microorganisms, (3) the early events of infection, which include rhizosphere-mediated attraction, adhesion and aggregation at the site of infection. We also draw some possible developments that would increase the understanding of the mechanisms underlying zoospores sensing and cellular responses.

# 2. The soil environment

## 2.1. Impact of soil on zoospores

As soil microswimmers, zoospores explore soil water films, air bubbles, humus, clay particles and mineral grains. They sense interfaces and surfaces, or bypass them to track their pathway toward the plant target (Fig. 2B,B' and Video S2). Water fluxes, combined with the soil microstructure and the autopropulsion capability of zoospores, are considered as significant contributors to disease outbreaks due to their influence on zoospore distribution. Zoospores move according to the microstructure composition comprising sand, clay or loam [21]. Soil particles create repulsion/attraction force fields affecting zoospore dissemination [22] depending on the capacity of negatively charged-soil particles to hold exchangeable cations. Nutrient cations absorbed by plants

**Fig. 1.** Structure and microswimmer traits of *Phytophthora* zoospores. (A) Micrograph of *P. parasitica* zoospore obtained using scanning electron microscopy (SEM). This shows the characteristic ellipsoidal zoospore cell body (Zcb) and the anterior and posterior flagella (Af and Pf, respectively). Tubular (left inset) and thinner (right inset) mastigonemes are found along the anterior flagellum, while the posterior flagellum is smooth. (B) Two-dimension schematic representation of the *P. palmivora* zoospore, including the two flagella beating with periodical waveforms in opposite directions and connected to the ellipsoidal cell body. The red arrows indicate the beating patterns of the flagella, while the blue arrow indicates the swimming direction of the zoospore. Cell body size and zoospore speed are obtained from Appiah *et al.* 2005 [9]. Panels C and C' show *P. parasitica* zoospores swimming in water (C) and the corresponding trajectory patterns delineated using the TrackMate plugin [19] as per the procedure detailed in Galiana *et al.* [15]. The trajectories indicate the randomness in swimming speed and direction of zoospores under no constraints. (C') Correspondence between colors and mean speed (µm/s) is indicated in the scale at the top of panel C'. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
<figure><img src="image_4.png" /></figure>

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<figure><img src="image_6.png" /></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 2.** Zoospore interactions with the surrounding environment. Panel A shows a schematic representation of a plant root being colonized by zoospores (Z). The zoomed longitudinal view highlights the different zones of the root tip (maturation zone (MaZ), elongation zone (EZ) and meristematic zone (MZ)), and illustrates the preferential aggregation of zoospores at the EZ as reported by Attard et al. [20]. Panel A' shows an EZ colonized by *P. parasitica* zoospores, 25 min after inoculation. Panels B, C and D give an overview of zoospore interactions with soil, plant and microbial environments, respectively. In Panel B, ionic signals emitted by charged soil particles and zoospore physical interactions with soil grains are represented. Panel B' shows a fluorescence micrograph of a sand grain surrounded by zoospores (Z) that are exploring its surface. For cytoplasmic staining, zoospores were initially loaded for 10 min with 1 µM BCECF-AM (2',7'-bis-carboxyethyl-5(6')-carboxyfluorescein acetoxymethyl ester). Panel C shows the ionic and chemical signals (e.g. root exudates) that are emitted or released by the plant root and subsequently attract zoospores. The fluorescence micrograph in C' shows *P. parasitica* BCECF-stained zoospores having colonized a tomato root in the soil. The profile of fluorescence (green) illustrates the complete coverage of a tomato root by emerging *Phytophthora* mycelium (the part which can be visualized among soil elements), as the result of an extensive colonization by zoospores a 90 min soil exploration. The vast majority of zoospores had reached the root while very few were dispersed or still exploring the soil microenvironment. Panel D shows mixed biofilm formation on the root surface with incorporation of bacteria (B) and newly attracted zoospores, was well as extracellular matrix (ECM) formation. Panel D' shows mixed biofilm formation on a tomato root surface. It illustrates the preferential colonization of the *Phytophthora* biofilm (Pb) rather than healthy root surface (hrs), by *Pseudomonas* species expressing Green Fluorescent Protein (GFP), 2 h post bacterial inoculation [18]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

are known to regulate the spatial abundances of soil bacterial
communities [23]. In their dissolved form, they appear to be a piv-
tal element in regulating zoospore release, motion and dissemina-
tion. Ca²⁺ treatments affect zoospore release (i) during cleavage of
the *P. parasitica* sporangium protoplasm into mono-nucleated cells
and (ii) when zoospores are released by dissolution of the spo-
rangium papillum [24]. *In vitro*, *P. cinamomi* zoospores exhibit
negative chemotaxis toward mono cations leading to collective
pattern formation [25,26]. K⁺ homeostasis influences the locomo-
tion and the encystment of zoospores. When K⁺ is applied as a gra-
dient, it provides guidance to *P. parasitica* zoospores and mediates
aggregation [15]. These results suggest that the diffusion of cations
in water films along the concentration gradient from soil particles
to plant roots contributes to shape microhabitats that are favorable
to *Phytophthora* zoospore dissemination and aggregate formation
in the soil.

Despite these advances, little is known about the factors con-
trolling zoospore behavior in porous media nor how these factors
contribute to the zoospore's preferential attraction to the root cues
of host plants. In order to produce disease models demonstrating
the incidence of a disease based on zoospore capability to reach
a host as a function of soil composition, a major challenge will be
the development of microfluidic devices to investigate zoospore
displacement in conditions designed to mimic the nature, the

geometry and the electric charges of soil particles [27,28]. Such
tools would also contribute to our understanding of how zoospores
sense and respond to ion stimuli, electric fields or physical obsta-
cles. Similar analyses could address zoospore behavior in different
types of soil microstructures after having loaded zoospores with
cellular probes (Fig. 2B' and Video S2) or using *Phytophthora* strains
expressing reporter genes encoding fluorescent proteins [29]. For
example, an agronomic challenge will be the analysis of soils char-
acterized by their exchange capacity of nutrient or metal cations
that are used to control oomycete diseases, such as Cu²⁺ in the case
of the Bordeaux Mixture. In such a case, the facilitated analysis of
zoospore displacement and distribution in soil, using labeled zoos-
pores to track them, should give information on how Cu²⁺-soil par-
ticles interactions impacting the metal retention capacity of the
soil may interfere with the management of disease dissemination.

## 2.2. Impact of zoospores on soil

*Phytophthora* species may play an important role in the soil where they decompose and recycle plant materials. For instance, in a study on microbial community functional structure variations under different soil management techniques, *P. cinamomi* polygalacturonase-expressed genes were found to be among the most abundant genes related to carbon degradation [30]. We know
very little about the specific roles of zoospores in ecological bal-
ance and their contribution may appear somewhat tenuous. How-
ever, the capacity of *Phytophthora* species to release 10⁵–10⁶
zoospores per infected plant in controlled conditions [16,31] is
indicative of their ability to induce radical change in the explored
soil environment. For example, the 30-fold upregulation of a gene
encoding a secreted alpha carbonic anhydrase (α-CA), observed in
*P. parasitica* zoospores upon aggregation establishment [32],
together with the oomycete's ability to produce a high-density
inoculum in proximity of the target [16], suggests a potential and
transitory role of this zoospore enzyme in non-photosynthetic
CO₂ fixation. A recent study showed that soil CA activity varied
with the diversity of microbial communities and CA gene expres-
sion patterns [33]. CA-mediated CO₂ hydration capacity was
assessed according to α-CA gene expression levels in algal and bac-
terial taxa, i.e. *Chlamydomonas reinhardtii* and Proteobacteria, even
though the expression patterns were difficult to interpret due to
the low representation of eukaryote metatranscriptomic data
[33]. This study pointed out the lack of data on environmental
CA distribution in lower eukaryotes, despite this being key to their
prominent role in CO₂ fixation. Interestingly, another study con-
ducted on karst ecosystems revealed higher CA activity in proxim-
ity to the soil surface and plant roots with higher extracellular CA
activity attributable to the fungal population, suggesting soil
eukaryotic microorganisms as an important source of CA activity
[34].

Expanding the *Phytophthora* representation in currently avail-
able genomic databases and developing metatranscriptomic and
enzymatic activity studies on soil micro-eukaryotic communities
[35–37] would contribute to the exploration of the potential role
of *Phytophthora* CAs in CO₂ fixation in the soil.

**3. The microbial environment**

3.1. *Zoospore-zoospore interactions*

An important question here is whether zoospores can sense the
difference between interactions amongst themselves and interac-
tions with the environment to eventually establish a collective
motion. Collective behaviors of zoospores have been described in
cell suspension, but remain poorly understood in general. Experi-
mental evidence has demonstrated that zoospore-zoospore inter-
actions can lead to "pattern swimming" in the absence of
chemical or electrical signals. Ochiai *et al.* showed that *P. citricola*
zoospores experience bioconvection pattern swimming in which
the zoospores swarm to a highly concentrated spot on the fluid
surface and then move downward and away from that spot due
to an increase in zoospore density and the depth of fluid [38]. This
suggests that zoospore-zoospore interactions are the result of
zoospore response to gyrotaxis. Additionally, Savory *et al.* con-
ducted experimental observations and proposed a mathematical
model revealing that, upon bioconvection in *P. infestans* zoospores,
chemotaxis leads to auto-aggregation of highly concentrated
plumes, which are advantageous in attacking local targets [39].
In *P. infestans* and *P. sojae*, the silencing of the G-protein α
subunit-encoding gene results in aberrant swimming patterns,
characterized by a higher frequency, sharp turns and shorter-
distance displacement compared to wild-type zoospores [40,41].
Moreover, as a consequence or as a concomitant effect of aberrant
motility, silencing of the same gene caused negative geotaxis (i.e.
attraction toward the surface), density-dependent auto-
aggregation and chemotaxis impairment in *P. infestans*, providing
a preliminary indication of the molecular pathways underlying
zoospore motility and collective behavior [40].

Nevertheless, these assays do not demonstrate what happens to
zoospores in soil, but rather suggest density instability with zoos-
pore motion being the determinant factor. Recent studies have
shown that *P. parasitica* zoospores display collective behavior and
*in vitro* aggregation patterns in response to a K⁺ external ionic gradi-
ent as a primary stimulus [13]. Aggregation is induced by a sequence
of events starting with negative chemotaxis, during which zoospores
move toward a region where the K⁺ concentration is < 1-4 mM,
resulting in upward zoospore migration and swarming. The
increased cellular density leads to bioconvection, with plume forma-
tion and downward migration, and consequent rapid aggregation
[15]. Investigations at the cellular and molecular level suggested
that this behavior could be regulated by cell-to-to cell signaling
and cation transport because Ca²⁺ and K⁺ channels were found to
be involved in K⁺ electroception and a remarkable K⁺-induced
enhancement of alpha carbonic anhydrase (α-CA) activity [32].

Additionally, previous studies showed that the perception and
the response to self-produced molecules determine *P. parasitica*
zoospore-zoospore communication and coordinated behavior, in a
way that is analogous to bacterial quorum sensing [42]. Zoospore-
secreted products stimulate cyst germination and induce a tactic
response to enhance zoospore auto-aggregation and infection estab-
lishment [42]. Nevertheless, the nature of these molecules and over-
all cellular responses that lead to coordinated behavior and
aggregation remain largely uncharacterized and require further
extensive characterization at both the cellular and molecular levels.

Zoospore-zoospore communication has also been proposed to
occur following the attraction process to a host. *P. parasitica*
appears to use such communication to amplify and increase local
adhesion by forming groups of cells that occupy specific or large
areas of the plant surface and undergo synchronized encystment
(Fig. 2D,D') [16]. The subsequent structure exhibits biofilm proper-
ties with mucin-like protein and polysaccharidic secretion [43],
cell-to-cell adhesion, self-produced matrix formation and constitu-
tion of channels used by still-swimming zoospores for exploration
[14]. The implication of the formation of such a structure on plant
infection remains to be fully established. It is possible that it cre-
ates a favorable environment for the exchange of signals and/or
nutrients between sessile, biofilm-associated cells and the zoos-
pores that are still swimming [16], and/or that mucins secreted
by zoospores and cysts have protective functions [44]. Another
question is whether biofilm formation occurs under natural condi-
tions, as is observed under laboratory conditions where zoospores
rapidly converge at the host root surface when these cells explore
porous soils (Video S3). Finally, although the molecular basis
remains to be determined, it should be noted that cross-talk
between zoospores of different species may occur at early stage
of infection. Supernatants conditioned by zoospores of four species
(*P. capsici*, *P. hydropathica*, *P. sojae*, and *P. nicotianae*) stimulate
infection on different host plants (*Catharanthus roseus*, *Lupinus
polyphyllus* and *Glycine max*) [45]

3.2. *Zoospore interactions with other microorganisms*

Zoospores explore the root environment together with the
other microorganisms living in the rhizosphere. The electrical sig-
naling mediated by bacterial ion channels that regulates the cell-
cell dialogue within bacterial biofilms [46] could also drive the
attraction/repulsion of neighboring zoospores through modifica-
tion of the membrane potential of zoospores. This could affect
the beating of the flagella or activate cellular responses, such as
osmoregulation. Chemotactic signaling pathways remain to be
characterized in this context.

Conversely, there is evidence showing that zoospore behavior in
the rhizosphere results in physical associations with a broad range
of microorganisms during root surface colonization (Fig. 2D'). This
contributes to the dissemination of *Phytophthora* propagules (Video S4) [47], the inhibition of zoospore movement and hyphal growth [48] or the colonization by other microorganisms, as illustrated in Fig. 2D' [17]. These findings, mainly descriptive, underscore the need to investigate how inoculum constitution on the host-root surface is affected by the microbial ecology of *Phytophthora*. In order to begin to delineate the *meta*-role of the microbial environment of *Phytophthora* species in the establishment of disease, studies based on 16S/18S and/or 26S rRNA sequencing have assessed microbial diversity in rhizospheric samples associated with *Phytophthora* infection [49] and compared it with that of healthy samples [18,49]. The analyses of *Quercus* spp. and *Curcubita* microbiomes established a positive correlation between the abundance of *Trichoderma* spp. and ectomycorrhizal fungi with a lower incidence of root disease caused by *Phytophthora* spp. [49,50]. Investigation of the rhizospheric bacterial microbiota associated with *P. parasitica* at the root surface of *Solanum lycopersicum* demonstrated a shift in the microbial community induced by *Phytophthora* infection, involving a Bacteroidetes/Proteobacteria transition with an enrichment of sequences assigned to the Bacteroidetes phylum and a reduction in those assigned to Proteobacteria [18]. Such resources also provide a basis to define the microbial inter-kingdom interactions regulating *Phytophthora* disease outcomes, and also those caused by bacteria. For instance, opportunistic *Pseudomonas* spp. establish commensal interactions with *P. parasitica*, preferentially colonizing the oomycete rather than the roots, so that they can infect plant cells [18]. By profiling the *A. thaliana* root microbiome, Durán *et al.* (2018) provided evidence that negative interactions between bacteria and oomycetes, members of root microbiota, are critical for plant survival and maintenance of the host-microbiota balance [51]. On the other hand, the rhizospheric microbiota of some wild *Solanum* species may contribute to off-season survival and pathogenicity of *P. infestans* [52]. In the specific context of zoospore swimming in the soil, such studies would shed light on how zoospores maximize microbial interactions during the early infection events to exploit the diversity of the effector repertoire that each species uses to promote infection [53,54]. The study of *Phytophthora* microbiota is also relevant to addressing the challenges associated with reducing pesticide use and developing bio-based materials for biocontrol and diagnostics [55].

## 4. The plant environment: The rhizosphere

The rhizosphere is the dynamic and heterogeneous soil space around the roots that is characterized by various connections between solid, liquid and gaseous substances and living species [36]. It has a pivotal role in plant growth promotion and nutrition [56]. The composition of the rhizosphere is mainly influenced by root soil acidification, H⁺ exchange, nutrient uptake and release of a wide range of exudates (sugars, polysaccharides, organic acids, sterols, phenolics, proteins, secondary metabolites and ions). Each of these compounds form a gradient across the rhizosphere along the longitudinal axis of the root. They are involved in attracting or repulsing beneficial and pathogenic microorganisms (Fig. 2C) [57,58,59]. Investigations addressing how and which rhizospheric compounds direct zoospore chemotaxis toward roots have resulted in the identification of stimuli among root exudates. For instance, *P. palmivora* is attracted by isovaleraldehyde, valeraldehyde and ante-isovaleraldehyde, compounds present in root exudates of many plants; *P. sojae* is attracted by isoflavones (daidzein, genistein) secreted by soybean roots [60]; and *Phytophthora* spp. are attracted by amino acids (aspartate, glutamate, asparagine, glutamine, arginine, methionine) secreted by many plants [8]. Ethanol is secreted by flooded roots and also attracts zoospores [60].

Despite molecular patterns governing chemotactic response remaining largely unknown, previous studies have reported the involvement of the G-protein signaling pathway in response to chemoattractants such as aspartate and glutamate in *P. infestans* [40], and daidzein and soybean roots in *P. sojae* [41,61,62].

Other studies have shown that *Phytophthora* zoospore motion is also driven by electric fields (electrotaxis) that are differentially produced by roots along their axes (Fig. 2C). *P. palmivora* presents an anodic taxis that drives zoospores to the rye grass root elongation zone (EZ) [14]. In different host species [14,20,63], zoospores preferentially aggregate at the root EZ (Fig. 2A,A') prior to penetration [20]. The EZ is the initial site of root cell growth where a shift to high rates of proton efflux generally begins and is controlled by the activity of plasma membrane proton pumps. This proton gradient contributes to the turgor pressure required to drive cell expansion and facilitate mineral nutrient uptake (e.g., K⁺, Na⁺, Ca²⁺, Mg²⁺, Cl⁻; [64]). Ionic exchanges and surface generated gradients associated with root growth in the EZ may play a crucial role in plant- *Phytophthora* interaction.

Thus, a number of root attractants and repellents for zoospores have been characterized. It is now important to define the parameters of effective chemoattraction at root surfaces. This will require characterization of the conditions that are necessary for the establishment of a stable gradient and chemoattraction. In this context, several parameters have to be defined: the spatio-temporal and concentration-scale of gradients at the root surface, the layer near the root in which gradients are stable [65], the zoospore distribution and the metrics of zoospore motion (velocity and trajectory) [15] in this environment. It is equally important to determine the genetic basis governing the release of attractants by the host plant. In particular, mutant screening strategies should be used to characterize the molecular actors (ions channels, transporters) and cellular mechanisms (secretory pathways, osmoregulation, nutrition of root cells, cell differentiation) regulating zoospore attraction. These studies will accelerate knowledge in the field of plant pathology, which has been somewhat overlooked in recent years, and could yield promising new targets for molecular breeding.

## 5. Concluding remarks

The versatile adaptive ability of zoospores to sense their environment is one of the key features of their evolutionary success in targeting host tissue. The availability of gene sequences and mRNA-level quantification data generated by the different *Phytophthora* genome projects has had a massive impact on the definition and the classification of the molecular repertoire at different stages of the *Phytophthora* life cycle, including zoospores [2]. Several studies have indicated the occurrence and the importance of putative pumps, ligand-gated channels, tyrosine kinase-like and G-protein signaling [32,66,67] in *Phytophthora*. Nevertheless, to date, very little is known about the major classes of receptor and signaling pathways involved in environment perception. The few available studies pointed out the role of a novel class of G-protein-coupled receptors (GPCRs) [61] and of the G-protein α subunit and its interacting protein, PsHint1 [41,62] in chemotaxis toward isoflavones and soybean roots, similarly to that observed for aspartate and glutamate in *P. infestans* [40]. To further advance this field, the next step will require a shift of focus toward the genetics and biochemistry of directional taxis mechanisms on both sides: the zoospore and plant cell.

The anterior flagellum is covered with mastigonemes and may constitute a nodal point to couple the chemical, electro- or mechanosensory pathways with the reconfiguration of the motor apparatus during different phases of motion. The asymmetric position of the two flagella with regard to the perception of a stimulus
during forward motion, and/or the variation in their plasma mem-
brane components could underpin the differential responses of
each flagellum to each kind of taxis [68]. To describe these mech-
anisms, the metrics of flagella beating need to be carefully
examined by high-speed camera analyses in microfluidic environ-
ments to mimic soil and plant surface compositions. The applica-
tion of microfluidics on zoospore research has not yet been fully
exploited, but is undoubtedly becoming indispensable.

A mathematical model is also needed to fully understand the
fundamentals behind the straight swimming trajectories, change
of direction, and zoospore-zoospore and zoospore-obstacle inter-
actions. Modeling methods of eukaryotic swimmers at low Rey-
nolds numbers usually require a solution for the Stokes equation
applied to flagellar motion to calculate the resultant cell body
movement [69]. Resistive force theory and slender-body theory
are the modeling methods most frequently used to predict forces
and movement. In the case of zoospores, the hydrodynamics of
an individual can be established by a simple microswimmer model
consisting of an ellipsoidal body and two flagella beating in period-
ical waveforms in opposite directions, to quantitatively character-
ize the activity of the two flagella and the propulsive efficiency
they produce. Moreover, a novel approach in quantifying the char-
acteristics of microswimmers is to exploit the universal distribu-
tions of their specific dynamical properties that are a
consequence of the variety of swimmer morphologies and sizes
[70]. Thus, the zoospore hydrodynamic model is important, as
we can derive the universal swimming speed of zoospores from
the characteristics of their flagella and bodies.

Further advancement of the research summarized here will pro-
vide information on the behavior of zoospores in the wild, and the
importance of soil reservoirs and environmental factors for sur-
vival, exploration, inoculum density and, finally, disease transmis-
sion. This will also deepen our understanding of the
epidemiological processes by which the abiotic and biotic environ-
ment affects plants infection by *Phytophthora* species and the sub-
sequent disease development.

CRediT authorship contribution statement

Ilaria Bassani: Writing - original draft, Visualization, Funding acquisition. Marie Larousse: Writing - original draft, Visualization.

Quang D. Tran: Writing - original draft, Visualization. Agnès

Attard: Writing - original draft, Visualization, Funding acquisition, Project administration. Eric Galiana: Writing - original draft, Visu-
alization, Funding acquisition, Project administration.

Declaration of Competing Interest

The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.

Acknowledgements

The authors acknowledge the CCMA (Centre Commun de Micro-
scopie Appliquée, Université Côte d'Azur, Microscopy and Imaging
platform Côte d'Azur, MICA) and its personnel. The SEM micro-
graphs shown in Fig. 1A were captured by François Orange. The
authors also thank the Microscopy Platform - Université Côte
d'Azur, INRAE, CNRS, ISA, France for the access to instruments
and technical advice. The authors acknowledge Abby Cutriss (office
of international scientific visibility, Université Côte d'Azur) and
William Rhamey for complete editing the manuscript.

We would like to thank the 4 reviewers and the editor for giving
valuable feedback to improve and extend the quality of the
manuscript.

This work has been supported by the French government
through the UCA<sup>EDI</sup> Investments in the Future project managed
by the National Research Agency with the reference number
ANR-15-IDEX-01; through the "Credits Scientifiques Incitatifs" of
the University of Nice Sophia-Antipolis and the "Action Recherche"
of the INRAE Plant Health and Environment Division.

The funders had no role in study design; in data collection, anal-
ysis and interpretation of data; in the writing of the manuscript;
and in the decision to submit the article for publication.

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