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The soil-borne white root rot pathogen Rosellinia necatrix expresses antimicrobial proteins during host colonization [1]

['Edgar A. Chavarro-Carrero', 'Laboratory Of Phytopathology', 'Wageningen University', 'Research', 'Wageningen', 'The Netherlands', 'Institute For Plant Sciences', 'Cluster Of Excellence On Plant Sciences', 'Ceplas', 'University Of Cologne']

Date: 2024-02

Rosellinia necatrix is a prevalent soil-borne plant-pathogenic fungus that is the causal agent of white root rot disease in a broad range of host plants. The limited availability of genomic resources for R. necatrix has complicated a thorough understanding of its infection biology. Here, we sequenced nine R. necatrix strains with Oxford Nanopore sequencing technology, and with DNA proximity ligation we generated a gapless assembly of one of the genomes into ten chromosomes. Whereas many filamentous pathogens display a so-called two-speed genome with more dynamic and more conserved compartments, the R. necatrix genome does not display such genome compartmentalization. It has recently been proposed that fungal plant pathogens may employ effectors with antimicrobial activity to manipulate the host microbiota to promote infection. In the predicted secretome of R. necatrix, 26 putative antimicrobial effector proteins were identified, nine of which are expressed during plant colonization. Two of the candidates were tested, both of which were found to possess selective antimicrobial activity. Intriguingly, some of the inhibited bacteria are antagonists of R. necatrix growth in vitro and can alleviate R. necatrix infection on cotton plants. Collectively, our data show that R. necatrix encodes antimicrobials that are expressed during host colonization and that may contribute to modulation of host-associated microbiota to stimulate disease development.

Most if not all organisms, including plants, associate with a wide diversity of microbes that live either inside these organisms, or in their immediate vicinity, and that collectively form their microbiota. Moreover, increasing evidence reveals that microbiota represent a key determinant for their health. To cause disease on their hosts, microbial pathogens need to overcome host immunity. Conceivably, pathogens need to overcome beneficial contributions that microbiota make to an organism’s health. Here, we show that the genome of the fungal white root rot pathogen Rosellinia necatrix encodes putatively secreted antimicrobial proteins, many of which are expressed during plant colonization. Two of these proteins are functionally analyzed in this study, and we reveal that they can inhibit the growth of bacteria that antagonise R. necatrix growth in vitro and that can alleviate R. necatrix infection on cotton plants. Thus, we propose that R. necatrix employs antimicrobials during host colonization to promote host infection through the selective manipulation of host microbiota.

Funding: EACC and DET acknowledge receipt of PhD fellowships from CONACyT, Mexico. ALM is holder of a postdoctoral research fellow funded by the 'Fundación Ramón Areces'. BPHJT acknowledges funding by the Alexander von Humboldt Foundation in the framework of an Alexander von Humboldt Professorship endowed by the German Federal Ministry of Education and Research, and is furthermore supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany´s Excellence Strategy – EXC 2048/1 – Project ID: 390686111. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2024 Chavarro-Carrero et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Similar to V. dahliae, also R. necatrix is a soil-borne pathogen that spends at least part of its life cycle in the soil, known to be an extremely competitive and microbe-rich environment [ 23 ]. In the present study, we aimed to generate a high-quality genome assembly to mine the R. necatrix genome for potential antimicrobial proteins that are exploited during host colonization and test the hypothesis that other pathogenic fungi besides V. dahliae exploit effector proteins with antimicrobial activity during host colonization as well.

It is generally accepted that plant pathogens secrete dozens to hundreds of so-called effectors into their host plants to stimulate host colonization [ 10 , 11 ]. Effectors can be defined as small, secreted proteins of ≤300 amino acids that are cysteine-rich and have tertiary structures that are stabilized by disulfide bridges and that are secreted by pathogens to promote disease on plant hosts [ 12 – 15 ]. However, also larger secreted proteins have been found to act as effectors, such as several LysM effectors [ 16 , 17 ]. Furthermore, also non-proteinaceous effectors have been described, such as fungal secondary metabolites as well as small RNA (sRNA) molecules that are delivered into host cells to suppress host immunity [ 18 ]. Many effectors have been shown to act in suppression of host immune responses [ 10 ]. However, recent studies have uncovered additional functions of pathogen effector proteins in host colonization [ 11 ]. For example, the soil-borne pathogen Verticillium dahliae secretes effector proteins to modulate microbiota compositions inside the host plant as well as outside the host in the soil to support host colonization [ 19 – 22 ]. One of them is the effector protein VdAve1 that was shown to display selective antimicrobial activity and facilitate colonization of tomato and cotton plants through host microbiota manipulation by suppressing antagonistic bacteria, such as members of the Spingomonadaceae [ 19 ]. Besides VdAve1, ten additional potentially antimicrobial effector protein candidates were predicted by mining the V. dahliae secretome for structural homologues of known antimicrobial proteins (AMPs) [ 19 ]. One of these candidates, VdAMP2, was subsequently found to contribute to V. dahliae survival in soil through its efficacy in microbial competition [ 19 ]. Another candidate, VdAMP3, was shown to promote microsclerotia formation in decaying host tissue through its antifungal activity directed against fungal niche competitors [ 20 ]. Most recently, a multiallelic gene homologous to VdAve1 was identified as VdAve1-like (VdAve1L) of which the VdAve1L2 variant was shown to encode an effector that promotes tomato colonization through suppression of antagonistic Actinobacteria in the host microbiota [ 22 ]. These findings have led to the hypothesis that microbiota manipulation is a general virulence strategy of plant pathogens to promote host colonization [ 21 ]. However, thus far evidence for such activity has been lacking for other fungal plant pathogens.

Various studies have addressed the biology of R. necatrix [ 6 , 7 ]. Nevertheless, the molecular mechanisms underlying pathogenicity of R. necatrix remain largely unexplored, mainly because the limited availability of genomic resources to date complicates investigations into the molecular biology of R. necatrix infections. Until recently, only a single Illumina technology short-read sequencing-based R. necatrix draft genome assembly was generated of strain W97 that was isolated from apple in Japan [ 8 ]. This 44 Mb genome assembly is highly fragmented as it comprises 1,209 contigs with 12,444 predicted protein-coding genes [ 8 ]. Recently, another assembly was released of a South-African strain (CMW50482) that was isolated from avocado, yet this assembly is even more fragmented with 1,362 contigs [ 9 ].

Plants infected by R. necatrix usually display two types of symptoms. The first type is displayed below-ground on the root system, where white and black colonies of mycelium can occur on the surface of infected roots. As the fungus penetrates and colonizes the root tissue, the roots acquire a dark brown color [ 5 ]. The second type of symptom occurs above-ground. These symptoms can develop rapidly as a consequence of the damaged root system and comprise wilting of leaves, typically after a period of drought or physiological stress, which affects plant vigor and eventually can lead to plant death. Symptoms of R. necatrix infection can also appear slowly, leading to a decline in growth, decreasing leaf numbers, along with wilting of leaves, chlorosis, and death of twigs and branches. On perennials these symptoms aggravate over time, and when moisture and temperature are unfavorable, the plant eventually dies [ 5 ].

Rosellinia necatrix is a prevalent soil-borne plant-pathogenic fungus that is found in temperate and tropical areas worldwide [ 1 ]. As causal agent of white root rot disease, R. necatrix has a broad host range comprising at least 170 species of dicotyledonous angiosperms that are dispersed over 63 genera and 30 families [ 2 ]. Many of these species are of great economic importance, such as Coffea spp. (coffee), Malus spp. (apple), Olea europea L. (olive), Persea americana Mill. (avocado), Prunus spp. (peaches, almonds, etc.), Vitis vinifera L. (grape) [ 3 ] and Rosa sp. (rose) [ 4 ].

Results

Absence of distinctive genome compartmentalization In many filamentous pathogens, effector genes are found in repeat-rich and gene sparse genomic compartments, whereas they are depleted in repeat-poor and gene-dense regions that typically harbor housekeeping genes, a genome organization that is typically referred to as the two-speed genome [55]. Thus, we aimed to explore if effector genes were associated with repetitive regions in R. necatrix too. Interestingly, the repeat content in R. necatrix strain R18 is low (2.39%) and evenly distributed throughout the genome, with the most abundant families being DNA/Helitron (67%) and long terminal repeats (LTRs; 31%). Accordingly, most repetitive elements are not preferentially located near effectors (p>0.05 after permutation test for distance). Moreover, effector genes are not preferentially found in regions with large intergenic distances (Fig 2). Thus, our results do not support association of effector genes with repeat-rich regions in R. necatrix. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Effector genes do not localize in gene-sparse regions. Gene density plot of ‘5 and ‘3 flanking log10 transformed intergenic distance with the dashed lines depicting the mean intergenic distances for all genes (A) and candidate effector genes encoded in the R. necatrix genome (B). https://doi.org/10.1371/journal.ppat.1011866.g002 Genomic comparisons in various filamentous fungi have revealed extensive chromosomal rearrangements and structural variants (SVs) in close association with effector genes [56–60]. Thus, we tested whether effector genes are associated with SVs in R. necatrix. First, to explore the genomic diversity among R. necatrix strains, a phylogenetic tree of all genomes was constructed with Realphy (version 1.12) [61]. Two clusters were identified, one with strains from Mexico isolated from roses, and one cluster with the strains from Spain and South Africa, isolated from avocado, as well as Japan, isolated from apple (Fig 3). Then, SVs were predicted for each of the genomes sequenced in this study using NanoSV (version 1.2.4 with default settings; [62]) by identifying split- and gapped-aligned long reads for the various strains to define breakpoint-junctions of structural variations when using the gapless genome assembly of strain R18 as a reference. We retrieved 2,639 SVs in total, comprising 1,264 insertions, 1,344 deletions, 4 inversions and 27 translocations (Fig 3A). The number of SVs per strain corresponds to their phylogenetic relationships based on whole-genome comparisons. To investigate the occurrence of the SVs in the R. necatrix strains, we calculated the frequency of each SV over the strains used in the analysis. Most of the SVs (94.2%) are shared by <50% of the strains, meaning that they occur in less than four strains. This suggests that SV is a common phenomenon in line with the phylogenetic and geographic relationship of R. necatrix strains. Interestingly, SVs occur all across the genome (Fig 3B), largely independently of repetitive regions (p>0.05 after permutation test for distance), but are found in close association with effector genes (p<0.05 after permutation test for distance). Collectively, our results for R. necatrix substantiate the lack of the typical genome compartmentalization that is associated with the two-speed genome organization that was found in many other filamentous fungi [63]. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Phylogeny and structural variation of nine Rosellinia necatrix strains. (A) Phylogeny of sequenced R. necatrix strains was inferred using Realphy [64]. The robustness of the phylogeny was assessed using 1000 bootstrap replicates, and branch lengths represent sequence divergence (branches with maximum (100%) bootstrap support are indicated in red). Rosellinia corticium was used to root the tree. The amount of structural variants was calculated using NanoSV [62] using strain R18 as a reference. (B) Circular plot displaying the genomic distribution of 2,019 SVs along the 10 chromosomes of R. necatrix. The tracks are shown in the following order from outside to inside: Gene density (10 kb), repetitive density (10 kb), effector gene locations, deletions, insertions and inversions and translocations. The color intensity of the lines for each SV track depict frequency in the R. necatrix collection. https://doi.org/10.1371/journal.ppat.1011866.g003

Weak structural clustering in the effector catalog Effectors continuously evolve towards optimal functionality while simultaneously evading recognition by plant immune receptors, which is considered one of the reasons for the lack of sequence conservation among effector proteins. Despite this lack of sequence conservation, groups of effector proteins that share their three-dimensional structure have been identified in various filamentous phytopathogens, such as the MAX-effector (Magnaporthe Avrs and ToxB like) family of the rice blast fungus Magnaporthe oryzae [65] that, together with some other families, are also found in the scab fungus Venturia inaequalis [66]. It has been speculated that MAX effectors exhibit this structural conservation as an adaptation either to the apoplastic environment, or for transport into the plant cytosol [65]. To investigate whether subsets of R. necatrix effectors display a similar fold conservation, their structures were predicted with Alphafold2 with an average quality score in a so-called predicted local distance difference test (pLDDT) of 86.1 (SD 11.8) on a scale from 0 to 100, with 100 indicating a perfect prediction. As only 11% of the predicted structures scored lower than 70 and only 2% lower than 50, we conclude that the fold prediction of the R. necatrix effector catalogue is generally robust. Next, we performed similarity clustering of the predicted effector folds, revealing that almost 40% of the effector candidates are structurally unique, while the remaining effectors could be assigned to a total of 31 clusters. As these clusters were only small, with on average only four members, we conclude that relatively little clustering occurs among R. necatrix effectors. To examine whether the observed clustering is merely based on structural similarity, or is mainly driven by sequence conservation, the five largest clusters that contain six to 11 members were further analysed (Fig 4B and 4C). Two of the five clusters show high average template modelling (TM) scores, with 0.85 and 0.91, respectively, on a scale from 0–1, while also showing a relatively high degree of sequence conservation of 43 and 47 percent, respectively. The other three clusters exhibit lower sequence conservation, with maximum 21 percent, but also show considerably lower respectively structural conservation, with TM scores between 0.57 and 0.64 only, indicating that structural similarity within the clusters is positively correlating with sequence conservation. Hence, the structural clustering can be explained by the sequence conservation amongst the effector proteins. Taken together, although a considerable number of structural effector clusters can be observed in R. necatrix, they contain only few members and structural conservation is mostly based on sequence conservation. Thus, effector family expansion seems to have played only a minor role in R. necatrix effector evolution. PPT PowerPoint slide

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TIFF original image Download: Fig 4. R. necatrix effector candidates show weak structural clustering, which is based on sequence conservation. (A) Ordered by hierarchical clustering based on structural similarity, the heatmap displays the structural similarity of each effector pair in an all-vs-all alignment based on template modelling (TM) scores that range from 0 to 1. Effector clusters were identified based on a similarity threshold > 0.5. The five largest clusters are highlighted with a black square and ordered by size. (B) Example structure for each of the five clusters based on the effector with the highest similarity to other effectors in the cluster. (C) Characteristics of the five largest structural effector clusters. https://doi.org/10.1371/journal.ppat.1011866.g004

Antimicrobial activity in culture filtrates It has recently been proposed that fungal plant pathogens employ effectors with antimicrobial activity to manipulate the host microbiota to promote infection [10,11,21]. To explore if the soil-borne fungus R. necatrix potentially exploits antimicrobials, we first tested whether R. necatrix culture filtrates can inhibit the growth of plant-associated bacteria. To this end, we collected R. necatrix culture medium after 4, 7 and 9 days of fungal growth by filtration through a 0.45 nm filter, and an aliquot of each of the culture filtrates was heat-inactivated at 95°C for 10 minutes. Finally, the culture filtrates were used as growth medium for individual bacterial species from a diversity panel of 37 plant-associated bacteria (S4 Table). After overnight incubation, growth of four of the 37 bacteria was inhibited in the 4- and 7-day culture medium filtrates when compared with cultivation in the heat-inactivated culture filtrates, namely Bacillus drentensis, Achromobacter denitrificans, Sphingobium mellinum and Flavobacterium hauense. While three of these were not found to be inhibited anymore in the 9-day culture medium filtrate, growth of B. drentensis was also still inhibited in this filtrate (Fig 5). Given that the heat-treatment inactivated the antimicrobial activity, suggesting that the activity is of proteinaceous nature, we passed the culture filtrates through a spin column with 3 kDa cut-off and tested the growth of Bacillus drentensis and Flavobacterium hauense in these filtrates. Interestingly, none of the filtrates inhibited bacterial growth, suggesting that the activity is mediated by proteins that are retained in the spin column, as metabolites and other small molecules are expected to pass through. Collectively, these findings suggest that the culture medium of R. necatrix contains (a) heat-sensitive protein(s) with selective antimicrobial activity. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Rosellinia necatrix culture filtrates inhibit the growth of particular plant-associated bacterial species. (A) Bacterial growth was measured over time in R. necatrix culture filtrates, which were obtained after 4 (red), 7 (grey) and 9 (green) days of fungal growth. Heat-inactivated culture filtrates collected after 4 (blue), 7 (yellow) and 9 (black) days of fungal growth were used as controls. (B) Growth of Bacillus drentensis and Flavobacterium hauense was measured in culture filtrates collected after 4 (red), 7 (grey) and 9 (green) days of fungal growth, and additionally in culture filtrates passed through spin-columns with 3 kDa cut-off after 4 (black), 7 (light green) and 9 (pink) days of fungal growth. The experiment was performed twice and error bars display standard deviations (n = 3). https://doi.org/10.1371/journal.ppat.1011866.g005

Several plant-associated bacteria display antagonistic activity One obvious reason for a fungus to target particular microbes with antimicrobials is that these may be detrimental to fungal growth due to the display of antagonistic activities. To test for such activities directed against R. necatrix, we conducted confrontation assays in vitro, where we grew R. necatrix near the individual bacteria species from the diversity panel of 37 bacteria (S4 Table). In these assays, we observed antagonistic effects of several bacterial species on R. necatrix (Fig 10). Interestingly, several of the bacterial species inhibited by either of the two effector proteins displayed antagonistic activity against R. necatrix, including Bacillus drentensis, Bacillus licheniformis, and Sphingobium mellinum (inhibited by FUN_011519), and Cellulosimicrobium cellulans, Chryseobacterium wanjuense and Pseudoxanthomonas suwonensis (inhibited by protein FUN_004580) (Fig 10). However, also some other bacterial species that were not found to be inhibited by either of the two effector proteins displayed antagonistic activity, namely Kaistia adipata, Pedobacter steynii, Pedobacter panaciterrae, Pseudomonas corrugata, Pseudomonas knackmusii, Solibacillus isronensis and Solibacillus silvestris. These findings suggest that a diversity of plant-associated bacteria possess inherent antagonistic activity against R. necatrix and that some of these bacteria are targeted by effector proteins of R. necatrix. PPT PowerPoint slide

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TIFF original image Download: Fig 10. Various plant-associated bacteria display antagonistic activity against Rosellinia necatrix. Fungal PDA disks were placed in the center of a petri dish and next to it individual bacteria species were deposited in a straight line with help of a spreader (n = 5), bacteria species were placed alone (right column) as controls. https://doi.org/10.1371/journal.ppat.1011866.g010

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