Unveiling the function and regulation control of the DUF3129 family proteins in fungal infection of hosts

Abstract

Many prokaryotic and eukaryotic proteins contain domains of unknown function (DUFs). A DUF3129 family of proteins is widely encoded in the genomes of fungal pathogens. A few studies in plant and insect pathogens indicated that the DUF3129 genes are required for fungal penetration of host cuticles with an unclear mechanism. We found that a varied number of DUF3129 proteins is present in different fungal species and the proteins are evolutionarily diverged from each other at the inter- and intra-specific levels. By using the insect pathogenic fungus Metarhizium robertsii as a model, we performed experiments and found that the seven DUF3129 proteins encoded by this fungus are localized to cellular lipid droplets (LDs). Individual deletion of these genes did not affect fungal formation of the infection structure appressoria and the accumulation of LDs in fungal conidia. When compared with the wild-type (WT) strain, insect bioassays revealed that the virulence of most null mutants were significantly impaired during topical infection but not during injection of insects. Carbon starvation and the subsequent Western blot analysis indicated that the LD-specific perilipin protein was completely degraded in the WT cells whereas varied levels of perilipin could be detected in the mutant cells, which signified that depletion of LD content was delayed in mutant cells, and DUF3129 proteins are therefore involved in LD degradation. We also provided biochemical evidence that these DUF3129 genes are transcriptionally regulated by a yeast Ste12-like transcription factor. The findings of this study not only unveil the function of DUF3129 proteins but also better understand the diverse mechanism of fungus–host interactions. This article is part of the theme issue ‘Biotic signalling sheds light on smart pest management’.

A M35 family metalloprotease is required for fungal virulence against insects by inactivating host prophenoloxidases and beyond

“…We found before that fungal infection could trigger insect cuticular immune responses as manifested by the formation of melanization spots [34,45]. Indeed, the formation of the black spots was observed during topical bioassays using the Galleria larvae (Figure 4(a)).…”

“…In brief, the 5ʹ-and 3ʹ-flanking sequences of each gene were amplified using the genomic DNA as a template with the Taq DNA polymerase (Vazyme, China) and different primers (Table S1). The fragments were purified, digested with restriction enzymes and then inserted into the corresponding restriction sites of the binary vector pDHt-Bar (conferring ammonium-glufosinate resistance) to produce the deletion vectors for Agrobacterium-mediated fungal transformation [34]. For complementation of MrM35-4 gene deletion, the full-length MrM35-4 gene including the promoter and 3ʹ-UTR region was amplified and cloned into the binary vector pDHt-Ben (conferring benomyl resistance) and the obtained plasmid was used to transform the null mutant ΔMrM35-4 for gene rescue.…”

“…Mortality was recorded every 12 h, and the median lethal time (LT 50 ) values were calculated by Kaplan-Meier analysis. During topical infection of the wax moth larvae, additional insects were treated for cuticular penetration and melanization comparison between WT and mutant infections [34].…”

“…The 10-fold dilution of cDNA was used for real-time RT-PCR analysis with a SYBR Mix (Toyobo, Japan). A β-tubulin gene was used as an internal reference [34]. The wax moth larvae injected with fungal spores (20 μl 5 × 10 6 conidia ml −1 per insect) for 36 h were used for fat body isolation and RNA extraction to detect the induction of the antifungal gallerimycin gene transcription in Galleria larvae [38].…”

Production of Diverse Beauveriolide Analogs in Closely Related Fungi: a Rare Case of Fungal Chemodiversity

“…After verification that C. militaris was nonvirulent to fruit flies, the WT strains of B. bassiana and B. brongniartii and besB and BrbesB deletion mutants were then assayed in parallel against the fruit flies and the last instar larvae of the wax moth G. mellonella as described before ( 40 ). Insect mortality was recorded every 12 h, and the survival dynamics were compared between the WT and mutant of each species by Kaplan-Meier analysis ( 47 ).…”