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

Abstract

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.

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

“…Leaves treated with water, DMSO/EtOH, MeOH, or toxin alone were used as mock controls. wall integrity in Δpex6 might be one of the reasons contributing to its lower viability (Wu et al, 2020a). The current study revealed that the pex6-deficient strain has lower chitin content of cell walls compared to wild-type.…”

“…Keissler used as a recipient host for transformation and mutagenesis experiments was obtained from a diseased citrus leaf and has been previously characterized ( Lin et al, 2009 ). Two pex6 mutant (Δ pex6 ) strains M4 and M9 were independently identified by targeted deletion of the Pex6 gene in wild-type ( Wu et al, 2020a ). The CP4 strain was identified by transforming and expressing a functional copy of Pex6 in Δ pex6 .…”

“…ACT was purified from culture filtrates of wild-type grown in PDB for 7 days using Amberlite XAD-2 resins (Sigma-Aldrich) and ethyl acetate as described ( Kohmoto et al, 1993 ). The quantity of ACT was calculated and normalized to the dry weight of mycelium as described previously ( Wu et al, 2020a ).…”

“…Deletion of the Pex6 gene homolog in the Japanese pear pathotype of A. alternata slightly reduces vegetative growth and conidiation, and completely shuts down the production of the host-selective AK toxin ( Imazaki et al, 2010 ). By contrast, deletion of the Pex6 homolog in the tangerine pathotype of A. alternata , which produces a host-selective toxin named Alternaria citri toxin (ACT), results in a severe growth reduction, causes a moderate reduction in toxin production, but has no effects on conidiation ( Wu et al, 2020a ). The pex6 -deficient mutant of the tangerine pathotype also reduces the formation of an appressorium-like structure (small, nonmelanized enlargement of hyphal tips), cell viability, and pathogenicity.…”

“…Because peroxisomes contain high levels of catalase ( Schrader and Fahimi, 2006 ; Antonenkov et al, 2010 ), we reasoned that peroxisomal functions might play a role in cellular resistance to ROS in the tangerine pathotype. However, mutational inactivation of Pex6 in this pathotype has no effects on ROS sensitivity ( Wu et al, 2020a ).…”