Untangling the responses of protistan communities associated with soil and plant compartments and their associations with bacterial and fungal communities to pathogen invasion are critical for understanding the ecological processes governing plant microbiome assembly. Here we examined the protistan communities across the soil–plant continuum of healthy chili peppers and those with Fusarium wilt disease (FWD) and integrated the bacterial and fungal microbiome data from our previous investigation in China. We found that FWD was associated with a significant enrichment of phagotrophic protists in roots and an increase in the proportion and connectivity of these phagotrophic protists in intra- and interkingdom networks. Specifically, FWD increased the negative correlations between phagotrophic protists (especially Cercozoa and Ciliophora) and several members of Actinobacteria, Alphaproteobacteria, and Gammaproteobacteria in the interkingdom networks. Furthermore, the microbiomes of diseased plants not only exhibit a higher relative abundance of functional genes related to bacterial anti-predator responses compared to healthy plants, but also contained a greater abundance of metagenome-assembled genomes possessing functional traits involved in this response. The increased microbial interkingdom correlations among bacteria, fungi, and protists, coupled with the enhanced effects of protists on bacteria and fungi, as well as the notable bacterial anti-predator feedback in the diseased plant microbiome, all suggest that FWD catalyzes the associations between different groups of microbiomes. These findings highlight the role of predatory protists in shaping microbial assembly and functionality through top-down forces during pathogenic stress, potentially contributing to co-evolution within these soil and plant microbiomes.

Rust fungi are characterized by large genomes with high repeat content, and have two haploid nuclei in most life stages, which makes achieving high-quality genome assemblies challenging. Here, we describe a pipeline using HiFi reads and Hi-C data to assemble a gigabase-sized fungal pathogen, Puccinia polysora f.sp. zeae, to haplotype-phased and chromosome-scale. The final assembled genome is 1.71 Gbp, with ~850 Mbp and 18 chromosomes in each haplotype, being currently the largest fungal genome assembled to chromosome scale. Transcript-based annotation identified 47,512 genes with a similar number for each haplotype. A high level of interhaplotype variation was found with 10% haplotype-specific BUSCO genes, 5.8 SNPs/kbp, and structural variation accounting for 3% of the genome size. The P. polysora genome displayed over 85% repeat content, with genome-size expansion, gene losses and gene family expansions suggested by multiple copies of species-specific orthogroups. Interestingly, these features did not affect overall synteny with other Puccinia species with smaller genomes. Fine-time-point transcriptomics revealed seven clusters of co-expressed secreted proteins that are conserved between two haplotypes. The fact that candidate effectors interspersed with all genes indicated the absence of a “two-speed genome” evolution in P. polysora. Genome resequencing of 79 additional isolates revealed a clonal population structure of P. polysora in China with low geographic differentiation. Nevertheless, a minor population drifted from the major population by having mutations on secreted proteins including AvrRppC, indicating the ongoing evolution and population differentiation. The high-quality assembly provides valuable genomic resources for future studies on the evolution of P. polysora.