Peroxisomes Implicated in the Biosynthesis of Siderophores and Biotin, Cell Wall Integrity, Autophagy, and Response to Hydrogen Peroxide in the Citrus Pathogenic Fungus Alternaria alternata

Abstract

Little is known about the roles of peroxisomes in the necrotrophic fungal plant pathogens. In the present study, a Pex6 gene encoding an ATPase-associated protein was characterized by analysis of functional mutations in the tangerine pathotype of Alternaria alternata, which produces a host-selective toxin. Peroxisomes were observed in fungal cells by expressing a mCherry fluorescent protein tagging with conserved tripeptides serine-lysing-leucine and transmission electron microscopy. The results indicated that Pex6 plays no roles in peroxisomal biogenesis but impacts protein import into peroxisomes. The number of peroxisomes was affected by nutritional conditions and H2O2, and their degradation was mediated by an autophagy-related machinery termed pexophagy. Pex6 was shown to be required for the formation of Woronin bodies, the biosynthesis of biotin, siderophores, and toxin, the uptake and accumulation of H2O2, growth, and virulence, as well as the Slt2 MAP kinase-mediated maintenance of cell wall integrity. Adding biotin, oleate, and iron in combination fully restored the growth of the pex6-deficient mutant (Δpex6), but failed to restore Δpex6 virulence to citrus. Adding purified toxin could only partially restore Δpex6 virulence even in the presence of biotin, oleate, and iron. Sensitivity assays revealed that Pex6 plays no roles in resistance to H2O2 and superoxide, but plays a negative role in resistance to 2-chloro-5-hydroxypyridine (a hydroxyl radical-generating compound), eosin Y and rose Bengal (singlet oxygen-generating compounds), and 2,3,5-triiodobenzoic acid (an auxin transport inhibitor). The diverse functions of Pex6 underscore the importance of peroxisomes in physiology, pathogenesis, and development in A. alternata.

Unveiling the Role Displayed by Penicillium digitatum PdMut3 Transcription Factor in Pathogen–Fruit Interaction

“…The involvement of peroxisomes in cell wall integrity has also been reported in other fungal pathogens such as Fusarium graminearum, M. oryzae, and A. alternata [45,[51][52][53]. As described above, in this study, deletant mutants showed a clear effect on cell wall integrity, which indicates that peroxisomes metabolism could be altered, affecting infection capacity.…”

“…Previous works showed that Mut3p protein has been associated with peroxisomes and methanol metabolism [40]. Multiple metabolic processes occur in peroxisomes, thus they play a crucial role in the development and pathogenesis of fungi and virulence [41][42][43][44][45]. For instance, peroxisomal metabolic function is required for appressorium-mediated plant infection by Colletotrichum lagenarium [42], peroxisomal fatty acid-oxidation occurred during plant infection in M. grisea [44], and peroxisome functions are of vital importance to pathogenesis of the tangerine pathotype of Alternaria alternata [45].…”

“…Multiple metabolic processes occur in peroxisomes, thus they play a crucial role in the development and pathogenesis of fungi and virulence [41][42][43][44][45]. For instance, peroxisomal metabolic function is required for appressorium-mediated plant infection by Colletotrichum lagenarium [42], peroxisomal fatty acid-oxidation occurred during plant infection in M. grisea [44], and peroxisome functions are of vital importance to pathogenesis of the tangerine pathotype of Alternaria alternata [45]. Taking all this together, it could be possible that PdMut3 plays an indirect role in fungal infectivity through control of metabolism, as deletion of this gene leads to increased infectivity.…”

Pexophagy is critical for fungal development, stress response, and virulence in Alternaria alternata

“…The WT strain of A. alternata used in this study has been previously characterized (Lin et al, 2009). WT expressing an mCherry fluorescent protein tagged with serine‐lysine‐leucine tripeptides (designated WT/mCherry‐SKL) was used to monitor the location of peroxisomes as previously described (Wu et al, 2021). Fungal strains were inoculated as a toothpick point on PDA (Difco) or MM (Chung et al, 2020) agar and incubated at 28°C.…”

“…To maintain proper cellular functions, cells have to keep intracellular ROS at a low level by removing excess ROS and oxidatively damaged components. Studies with A. alternata have demonstrated that the number of peroxisomes decreases upon exposure to environmental H 2 O 2 (Wu et al, 2021), suggesting that H 2 O 2 may induce the turnover of peroxisomes via pexophagy, a selective autophagic pathway involved in the degradation of peroxisomes in vacuoles. Peroxisomes are membrane‐enclosed microbodies engaged in a number of metabolic functions in eukaryotic cells (Okumoto et al, 2020).…”