DATA AVA I L A B I LIT Y S TATEMENT
The protistan amplicon sequencing data have been submitted to the NCBI Sequence Read Archive (SRA) database under the accession number PRJNA680484. Previous data that support the findings of this study had been deposited in SRA under the accession number PRJNA667302 (16S), PRJNA667299 (ITS), and PRJNA667562 (Metagenomic). The scripts used for computational analyses and plotting figures are available at https://github.com/MinGao1/Min_protist_2023.git.

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FIGURE LEGENDS

FIGURE 1 Assembly of the protistan communities in pepper. aNon-metric multi-dimensional scaling (NMDS) ordinations of Bray–Cutis dissimilarity matrices with permutational analysis of variance (PERMANOVA). NMDS and PERMANOVA in each compartment can be found in Figure S1. b Contribution of FWD and sampling site to the variation of protistan community in each compartment, based on PERMANOVA. c Volcano plots illustrating the enrichment and depletion of protistan ZOTUs in the diseased organs compared with the healthy. Functional group of these enriched or depleted protistan ZOTUs appears in Figure S3.
FIGURE 2 Protistan intra-kingdom co-occurrence networks. aProtistan intra-kingdom networks showing a higher number of nodes and edges in diseased networks than in the healthy. The nodes are colored according to protistan lineages, and the size of node indicates the degree of correlations. The edges are colored to indicate the positive (green) and negative (red) correlations. b Locations of differentially abundant ZOTUs and core ZOTUs are labeled in the healthy and diseased networks. Purple and orange colors of nodes indicate differentially abundant ZOTUs and core ZOTUs, respectively. Nodes with yellow asterisks represent phagotrophic protists. Numbers and degree of these differentially abundant ZOTUs and core ZOTUs are shown for the healthy (c ) and diseased (d ) networks. eComparison of node-level topological features in Figure 2a(degree and closeness centrality) demonstrating that more nodes with a high degree (i.e., > 50) were recorded in the diseased than healthy network. f Degree of Cercozoa in healthy and diseased networks. Significance differences were determined by nonparametric Kruskal–Wallis test. g Degree of Cilicophora in healthy and diseased networks. Significance differences were determined by nonparametric Kruskal–Wallis test.
FIGURE 3Interkingdom co-occurrence networks .a Networks that integrated bacterial, fungal, and protistan ZOTUs, showing a higher number of fungal taxa (orange color) and protistan taxa (yellow color), and a lower number of bacterial taxa (blue color) in diseased network than those in its healthy network. b Comparison of node-level topological features in Figure 3a (degree and closeness centrality).c Numbers of bacterial–bacterial (BB), bacterial–fungal (BF), fungal–fungal (FF), bacterial–protistan (BP), fungal–protistan (FP), and protistan–protistan (PP) correlations in the healthy and diseased networks. Green or red coloring of a given column indicate a positive or negative correlation, respectively. d Networks constructed after filtering to retain the nodes of Cercozoa and Ciliophora and related nodes and links in the healthy and diseased network. Taxonomic information for these nodes is conveyed in Figure S10. eNumbers of correlations between Cercozoa/Ciliophora and other members, including Actinobacteria, Alphaproteobacteria, Gammaproteobacteria, Sordariomycetes, Cercozoa, Ciliophora, and Discoba in the healthy and diseased networks. f Positive correlations between Cercozoa/Ciliophora and bacteria; note the words of potential beneficial bacteria written in red. The coloring of the nodes is consistent with Figure 3d.
FIGURE 4 Assembly processes of protistan, bacterial, and fungal microbiomes and functional genes related to prey defense on the microbiome of pepper plants. a Relative contribution of determinism and stochasticity on protistan, bacterial, and fungal community assembly process between healthy and diseased plants based on the β-Nearest Taxon Index (βNTI) values. D means deterministic process, S means stochastic process. b Relative contribution of five ecological processes on bacterial, fungal, and protistan microbiome assembly between healthy and diseased plant. c Box plots showing the proportion of functional genes linked to prey defense traits (hydrogen cyanide, cyclic lipopeptides, and type III secretion systems) on the pepper root endosphere microbiomes.
FIGURE 5 Functional genes related to bacterial predator defense discovered in the metagenome-assembled genomes (MAGs). a Phylogenetic tree of 56 bacterial MAGs recovered from the root endosphere microbiomes, including 15 and 41 MAGs recovered from healthy root (named “H*”) and diseased root (“D*”) samples, respectively. The completeness, contamination and classification of these recovered bacterial MAGs are given in Table S6. b Heat map for the abundance of functional genes (based on KO) related to prey defense traits to (hydrogen cyanide, cyclic lipopeptides, and type III secretion systems) from all the recovered MAGs. c Abundance of MAGs that carry predator defense functional genes on the microbiome of healthy and diseased plants.
FIGURE 6 Schematic drawing depicting effects of pathogen invasion on pepper microbiome assembly. Infection of pepper plants by FWD results in the enhancement of protist–prey interactions, prey defense traits, and determinism. A comprehensive understanding of microbe–microbe interactions under pathogen invasion is generated, especially emphasizing the ecological importance of the top-down forces by protists. This figure was created by Biorender.