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.