The endoparasitic fungus Hirsutella rhossiliensis is an important biocontrol agent of cyst nematodes in nature. To determine the potential parasitism of the fungus on a non-natural host, the pinewood nematode (Bursaphelenchus xylophilus) living in pine trees and the endophytic ability of the fungus on plants, in this paper, we first constructed and utilized a green fluorescent protein (GFP)-tagged H. rhossiliensis HR02 transformant to observe the fungal infection process on B. xylophilus and its colonization on Arabidopsis roots. Then, we compared the fungal parasitism on three species of nematodes with different lifestyles, and we found that the fungal parasitism is correlated with nematode species and stages. The parasitic effect of H. rhossiliensis on adults of B. xylophilus is similar to that on second-stage juveniles (J2) of the root-knot nematode Meloidogyne incognita after 24 h of inoculation, although the virulence of the fungus to second-stage juveniles of M. incognita is stronger than that to those of B. xylophilus and Caenorhabditis elegans. Moreover, the endophytism of H. rhossiliensis was confirmed. By applying an appropriate concentration of H. rhossiliensis conidial suspension (5 × 10⁶ spores/mL) in rhizosphere soil, it was found that the endophytic fungus can promote A. thaliana growth and reproduction, as well as improve host resistance against M. incognita. Our results provide a deeper understanding of the fungus H. rhossiliensis as a promising biocontrol agent against plant-parasitic nematodes.

An effective selection marker is necessary for genetic engineering and functional genomics research in the post-genomic era. Isaria javanica is an important entomopathogenic fungus with a broad host range and prospective biocontrol potentials. Given that no antibiotic marker is available currently in this fungus, developing an effective selection marker is necessary. In this study, by applying overlap PCR and split-marker deletion strategy, combining PEG-mediated protoplasm transformation method, the uridine auxotrophy gene (ura3) in the I. javanica genome was knocked out. Then, using this transformation system, the pH response transcription factor gene (IjpacC) was disrupted successfully. Loss of IjpacC gene results in an obvious decrease in conidial production, but little impact on mycelial growth. The virulence of the ΔIjpacC mutant on caterpillars is similar to that of the wild-type strain. RT-qPCR detection shows that expression level of an acidic-expressed S53 gene (IF1G_06234) in ΔIjpacC mutant is more significantly upregulated than in the wild-type strain during the fungal infection on caterpillars. Our results indicate that a markerless transformation system based upon complementation of uridine auxotrophy is successfully developed in I. javanica, which is useful for exploring gene function and for genetic engineering to enhance biological control potential of the fungus.

Peptidases are very important to parasites, which have central roles in parasite biology and pathogenesis. In this study, by comparative genome analysis, genome-wide peptidase diversities among plant-parasitic nematodes are estimated. We find that genes encoding cysteine peptidases in family C13 (legumain) are significantly abundant in pine wood nematodes Bursaphelenchus genomes, compared to those in other plant-parasitic nematodes. By phylogenetic analysis, a clade of B. xylophilus-specific legumain is identified. RT-qPCR detection shows that these genes are highly expressed at early stage during the nematode infection process. Utilizing transgene technology, cDNAs of three species-specific legumain were introduced into the Arabidopsis γvpe mutant. Functional complementation assay shows that these B. xylophilus legumains can fully complement the activity of Arabidopsis γVPE to mediate plant cell death triggered by the fungal toxin FB1. Secretory activities of these legumains are experimentally validated. By comparative transcriptome analysis, genes involved in plant cell death mediated by legumains are identified, which enrich in GO terms related to ubiquitin protein transferase activity in category molecular function, and response to stimuli in category biological process. Our results suggest that B. xylophilu-specific legumains have potential as effectors to be involved in nematode-plant interaction and can be related to host cell death.

Proteins containing the SQS-PSY domain, which include squalene synthetase (SQS), phytoene synthetase (PSY), and NDUFAF6, are functionally important and widely distributed in plants and animals. However, they have not been previously reported in nematodes. In this study, we identified a gene (Minc31999) encoding an SQS-PSY domain-containing protein in the root-knot nematode Meloidogyne incognita. In silico comparison and enzymatic assays of the recombinant protein indicated that this nematode protein is a putative NDUFAF6 homolog. Phylogenetic analysis revealed that this protein is evolutionarily conserved within the Nematoda phylum. RT-qPCR analysis showed that Minc31999 is highly expressed during the early infection stage of M. incognita. Targeting the nematode gene Minc31999 via host-induced gene silencing (HIGS) significantly hindered nematode development and virulence. In contrast, heterologous expression of Minc31999 in Arabidopsis thaliana disrupted normal plant development and increased host susceptibility to nematode infection. Transcriptomic profiling (RNA-seq) of these transgenic plants prior to infections showed a widespread differential expression of genes across multiple metabolic pathways. We propose that this nematode SQS-PSY domain-containing protein may function as an effector that rewires host secondary metabolism to establish a parasitic relationship. Our study elucidates a novel strategy in nematode–plant interactions and advances our understanding of the functional evolution of SQS-PSY domain-containing proteins.