The pre‐rRNA processing factor Nop53 regulates fungal development and pathogenesis via mediating production of reactive oxygen species

Abstract

Botrytis cinerea is a necrotrophic plant fungal pathogen that annually causes enormous economic losses worldwide. The ribosome is an organelle for cellular protein biosynthesis. However, little is known about how the ribosome operates as a machine to mediate microbial pathogenesis. Here, we demonstrate that Nop53, a late-acting factor for 60S ribosomal subunit maturation, is crucial for the pathogen's development and virulence. BcNop53 is functionally equivalent to yeast nop53p. Complementation of BcNOP53 completely restored the growth defect of the yeast Δnop53 mutant. BcNop53 is located in nuclei and disruption of BcNOP53 also dramatically impaired pathogen growth. Deletion of BcNOP53 blocked infection structure formation and abolished virulence of the pathogen, possibly due to reduced production of reactive oxygen species. Moreover, loss of BcNOP53 impaired pathogen conidiation and stress adaptation, altered conidial and sclerotial morphology, retarded conidium and sclerotium germination as well as reduced the activities of cell-wall degradation-associated enzymes. Sclerotium production was, however, increased. Complementation with the wild-type BcNOP53 allele rescued defects found in the ΔBcnop53 mutant. Our work establishes a systematic elucidation of Nop53 in regulating microbial development and pathogenesis, provides novel insights into ribosomal processes that regulate fungal pathogenesis, and may open up new targets for addressing fungal diseases.

Transcriptome analysis and functional validation reveal a novel gene, BcCGF1, that enhances fungal virulence by promoting infection‐related development and host penetration

“…DAB is converted to dark-brown polymers in the presence of H 2 O 2 and NBT forms a dark-blue water-insoluble formazan precipitate on reduction by superoxide radicals. We also determined the influence of cAMP signalling on conidial germination and/or IFS formation of the tested strains as previously described (Feng et al, 2017;Cao et al, 2018;Hou et al, 2020). Our data demonstrate that the ∆Bccgf1 mutants are difficult to be stained in the germ-tubes and growing mycelia during conidial germination, appressorium, and infection structure formation (Figures 8a and S7); quantification analysis indicated that the relative ROS production by the mutants was significantly reduced (Figure 8b).…”

“…Two P450 monooxygenase genes (BCIN_07g05430 and BCIN_03g06490) involved in ABA biosynthesis in B. cinerea were upregulated (Table S5). NADPH oxidases are required for ROS production and are involved in conidial germination, differentiation, and vegetative and pathogenicity development as well as virulence in B. cinerea (Marschall and Tudzynski, 2016a;Cao et al, 2018;Hou et al, 2020). NADPH oxidase genes BcNOXA (BCIN_05g00350) and…”

“…The mutant strains displayed reductions in conidial germination and subsequent hyphal growth (Figure 7a,c) and reductions in radial growth of mycelia on CM containing the assorted stress agents (Figure 7b,d). Further analysis indicated that the relative mycelial radial growth inhibition (Cao et al, 2018;Hou et al, 2020) of the ΔBccgf1 strains significantly increased when cultured on CM containing the stress-mimicking agents compared to that of the control and complemented strains (Figure 7e,f). These data suggest that BcCGF1 is required for pathogen osmotic and oxidative stress adaptation as well as cell wall integrity.…”

“…Data represent means ± SD from at least four independent experiments. **, ***Significance at p < .01 and p < .001, respectively nitroblue tetrazolium (NBT) staining approaches (Cao et al, 2018;Hou et al, 2020). DAB is converted to dark-brown polymers in the presence of H 2 O 2 and NBT forms a dark-blue water-insoluble formazan precipitate on reduction by superoxide radicals.…”

“…The interactions of the autophagy proteins F I G U R E 8 BcCGF1 regulates endogenous reactive oxygen species (ROS) production in Botrytis cinerea and cAMP rescues the infectionrelated development in the BcCGF1 deletion strains. Conidia of the wild type (B05.10), ∆Bccgf1, and ∆Bccgf1-C strains were cultivated on complete medium (CM) or CM supplemented with diphenylene iodonium (DPI) or dithiothreitol (DTT) at 20 °C for the indicated lengths of time, and then the germ tubes or hyphae/mycelia of the tested strains were stained with 3,3′-diaminobenzidine (DAB) or nitroblue tetrazolium (NBT) solution as previously described (Cao et al, 2018;Hou et al, 2020). (a) Disruption of BcCGF1 reduced ROS production in the ∆Bccgf1 mutants during infection-related development detected by DAB or NBT staining.…”