The Pex3-mediated peroxisome biogenesis plays a critical role in metabolic biosynthesis, stress response, and pathogenicity in Alternaria alternata.

Peroxisomes are microbodies involved in the metabolism of fatty acids and hydrogen peroxide (H2O2) in eukaryotes. In the current study, an AaPex3 gene encoding a peroxisome membrane protein was demonstrated to be required for peroxisome biogenesis and resistance to peroxides and superoxide-generating compounds. Deleting AaPex3 affected the expression of the genes encoding the NADPH oxidase (NoxA) and the Yap1 stress-responsive transcription regulator, both of which have been implicated in ROS resistance. The AaPex3-mediated peroxisome biogenesis negatively affected resistance to singlet oxygen-generating compounds, 2-chloro-5-hydroxypyridine (CHP), and 2,3,5-triiodobenzoic acid (TIBA), novel phenotypes associated with peroxisomes. Nile red staining revealed that ΔAaPex3 accumulated more lipid bodies than the wild type. ΔAaPex3 conidia had thinner cell walls than the wild type, suggesting the involvement of AaPex3 in maintaining cell wall integrity. Genetic evidence has also demonstrated that the AaPex3-mediated peroxisome biogenesis is required for conidiogenesis, conidia germination, siderophore biosynthesis, toxin production, and virulence. Biotin or lipids could restore ΔAaPex3 growth in axenic culture and on the surface of citrus leaves. In contrast, co-application of ΔAaPex3 with biotin and oleic acid on citrus leaves failed to induce necrotic lesions. Our results revealed the multifaceted functions of peroxisomes in the phytopathogenic fungus.

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

Alternaria alternata can resist high levels of reactive oxygen species (ROS). The protective roles of autophagy or autophagy-mediated degradation of peroxisomes (termed pexophagy) against oxidative stress remain unclear. The present study, using transmission electron microscopy and fluorescence microscopy coupled with a GFP-AaAtg8 proteolysis assay and an mCherry tagging assay with peroxisomal targeting tripeptides, demonstrated that hydrogen peroxide (H2 O2 ) and nitrogen depletion induced autophagy and pexophagy. Experimental evidence showed that H2 O2 triggered autophagy and the translocation of peroxisomes into the vacuoles. Mutational inactivation of the AaAtg8 gene in A. alternata led to autophagy impairment, resulting in the accumulation of peroxisomes, increased ROS sensitivity, and decreased virulence. Compared to the wild type, ΔAaAtg8 failed to detoxify ROS effectively, leading to ROS accumulation. Deleting AaAtg8 down-regulated the expression of genes encoding an NADPH oxidase and a Yap1 transcription factor, both involved in ROS resistance. Deleting AaAtg8 affected the development of conidia and appressorium-like structures. Deleting AaAtg8 also compromised the integrity of the cell wall. Reintroduction of a functional copy of AaAtg8 in the mutant completely restored all defective phenotypes. Although ΔAaAtg8 produced wild-type toxin levels in axenic culture, the mutant induced a lower level of H2 O2 and smaller necrotic lesions on citrus leaves. In addition to H2 O2 , nitrogen starvation triggered peroxisome turnover. We concluded that ΔAaAtg8 failed to degrade peroxisomes effectively, leading to the accumulation of peroxisomes and the reduction of the stress response. Autophagy-mediated peroxisome turnover could increase cell adaptability and survival under oxidative stress and starvation conditions.

Proper Functions of Peroxisomes Are Vital for Pathogenesis of Citrus Brown Spot Disease Caused by Alternaria alternata.

In addition to the production of a host-selective toxin, the tangerine pathotype of Alternaria alternata must conquer toxic reactive oxygen species (ROS) in order to colonize host plants. The roles of a peroxin 6-coding gene (pex6) implicated in protein import into peroxisomes was functionally characterized to gain a better understanding of molecular mechanisms in ROS resistance and fungal pathogenicity. The peroxisome is a vital organelle involved in metabolisms of fatty acids and hydrogen peroxide in eukaryotes. Targeted deletion of pex6 had no impacts on the biogenesis of peroxisomes and cellular resistance to ROS. The pex6 deficient mutant (Δpex6) reduced toxin production by 40% compared to wild type and barely induce necrotic lesions on citrus leaves. Co-inoculation of purified toxin with Δpex6 conidia on citrus leaves, however, failed to fully restore lesion formation, indicating that toxin only partially contributed to the loss of Δpex6 pathogenicity. Δpex6 conidia germinated poorly and formed fewer appressorium-like structures (nonmelanized enlargement of hyphal tips) than wild type. Δpex6 hyphae grew slowly and failed to penetrate beyond the epidermal layers. Moreover, Δpex6 had thinner cell walls and lower viability. All of these defects resulting from deletion of pex6 could also account for the loss of Δpex6 pathogenicity. Overall, our results have demonstrated that proper peroxisome functions are of vital importance to pathogenesis of the tangerine pathotype of A. alternata.

Biotin biosynthesis affected by the NADPH oxidase and lipid metabolism is required for growth, sporulation and infectivity in the citrus fungal pathogen Alternaria alternata.

The tangerine pathotype of Alternaria alternata affects many citrus cultivars, resulting in yield losses. The capability to produce the host-selective toxin and cell-wall-degrading enzymes and to mitigate toxic reactive oxygen species is crucial for A. alternata pathogenesis to citrus. Little is known about nutrient availability within citrus tissues to the fungal pathogen. In the present study, we assess the infectivity of a biotin deficiency mutant (ΔbioB) and a complementation strain (CP36) on citrus leaves to determine how biotin impacts A. alternata pathogenesis. Growth and sporulation of ΔbioB are highly dependent on biotin. ΔbioB retains its ability to acquire and transport biotin from the surrounding environment. Growth deficiency of ΔbioB can also be partially restored by the presence of oleic acid or Tween 20, suggesting the requirement of biotin in lipid metabolism. Experimental evidence indicates that de novo biotin biosynthesis is regulated by the NADPH oxidase, implicating in the production of H2O2, and is affected by the function of peroxisomes. Three genes involved in the biosynthesis of biotin are clustered and co-regulated by biotin indicating a transcriptional feedback loop activation. Infectivity assays using fungal mycelium reveal that ΔbioB cultured on medium without biotin fails to infect citrus leaves; co-inoculation with biotin fully restores infectivity. The CP36 strain re-expressing a functional copy of bioB displays wild-type growth, sporulation and virulence. Taken together, we conclude that the attainability or accessibility of biotin is extremely restricted in citrus cells. A. alternata must be able to synthesize biotin in order to utilize nutrients for growth, colonization and development within the host.