Metagenomic dynamics in Olea europaea after root damage and Verticillium dahliae infection

The olive tree is of particular economic interest in the Mediterranean basin. Researchers have conducted several studies on one of the most devastating disorders affecting this tree, the Verticillium wilt of olive, which causes significant economic damage in numerous areas of this crop. We have analyzed the temporal metagenomic samples of a transcriptomic study in Olea europaea roots and leaves after root-damage and after a root Verticillium dahliae infection (Jimenez-Ruiz et al. 2017). Our results indicate that this infection, although led by Verticillium, is driven not by a single species but by a polymicrobial community, including their natural endophytes, which acts as a consortium in the attack to the host plant. This community includes both biotrophic and necrotrophic organisms that alternate and live together during the infection. Our results not only describe how the microbial community progresses along these processes, but also explain the high complexity of these systems, that in turn, could justify at least in part the occasional changes and disparity found at the time of classifying the kind of parasitism of a determined organism.


Introduction
The olive tree could be the earliest cultivated temperate fruit since paleobotanists have traced back its do-27 mestication to the early Neolithic age (Terral and Arnold-Simard 1996). At present, both the cultivation of 28 olive and the olive-oil related industry have grown to the point of having a profound worldwide socioeconomic 29 and environmental impact. 30 As the Verticillium wilt of olive is one of the most devastating disorders affecting this crop, a transcriptomic 31 RNA-seq analysis was recently conducted to study the interaction between Olea europaea and Verticillium 32 dahliae (Jimenez-Ruiz et al. 2017), which concluded that mainly a ROS response appeared first in the pathogen 33 and later in the plant. 34 In recent years, there has been a deviation from studying individual species to study the whole community 35 that is actually living in a determined niche. Shotgun metagenomic sequencing (SMS) helps this paradigm 36 shift by the use of massive nucleic acid sequencing of whole metagenomes obtained from samples with the 37 extra advantage of not requiring prior knowledge of the species that are present.

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The description of these interactions are, however, variable and somehow confusing in many cases. So, V. 42 dahliae has been described as an hemi-biotrophic pathogen because it seems to behave as biotrophic during bases underlying these different parasitical alternations are still not fully understood. 47 We must emphasize that the use of metagenomic data dealing with the process of a fungal plant infection     in the sequenced reads evolved from a unimodal distribution peaking at 43%, coincident with that of the 94 olive genome, to a pseudogaussian distribution reflecting a higher GC content ratio (53%) 15-day after the 95 inoculation. Figures 1b and 1c show the percentage of reads coming from infected roots mapped either with 96 Kallisto or STAR to the olive genome drastically decreasing along the infection. The number of Verticillium 97 reads was very low in the infected samples. As expected, Verticillium reads were negligible or missing in the 98 controls and leaves. However, the per base GC content and the proportion of mapped reads in the leaf samples 99 to the olive genome remained practically constant. All these data taken together confirmed that the infection 100 progressed through the roots and that there was a progressive emergence of other biological organisms during 101 infection that were displacing the olive tree in terms of mRNA abundance. The species origin of the unmapped 102 reads was unknown, and thus, a metagenomic analysis was required.          is also able to prey on larger nematodes thanks to efficient and specialized cooperative hunting (Geisen et al.

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The fungi Rhizoctonia solani, R. sp. AG-Bo, and Ceratobasidium sp. AG-A belongs to the same cluster 202 (see Figure 3). As we can see in Figure 2, these fungi presented a very low frequency of mapped reads during cosmopolitan nematode species in soil (Félix 2006     Nevertheless, other taxa, at the end of the process (7 days), recovered a rank similar to the initial one.

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That is the case of the plant pathogen Phytophthora sojae, which causes root rot of soybean. P. sojae had     We see that Taylor's power law seems to be ubiquitous, spanning in this case more than six orders of magnitude.

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Our results suggest that this disease, although led by Verticillium, is driven not by a single species but by a 284 polymicrobial community which acts as a consortium in the attack of another community formed by the host 285 plant and its endophytes, as Figure 9 shows. Thus, an infectious process can be generalized with a systems 286 biology approach as an attack of one system, i.e. a polymicrobial community, to another, the host and its 287 symbionts. Indeed, our systems approach to the Verticillium wilt of olive has revealed a complex interaction 288 between complex systems.

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Longitudinal (temporal) metagenomic analysis of the shotgun metagenomic sequencing data has allowed 290 us to study the overall dynamics of the system as well as to obtain results split into amoebae and ciliates, 291 fungi, bacteria, and nematodes. Besides, the longitudinal analysis of a root damage process has served as a 292 real "dynamic control dataset" of the infection process afflicting the olive rhizosphere.

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Our results also have very important implications in relation to the assignation of a determined parasitic 294 species as biotroph, necrotroph or hemibiotroph. Verticillium, for example, has been sometimes defined as a 295 biotrophic fungus (Boogert and Deacon 1994), whereas some other studies define it as hemibiotrophic (Zhou 296 bioRxiv pre-print L A T E X v0.1          In the bottom of the pie, Candida albicans appears with a good average score but low frequency.