Functional Analysis of Autophagy-Related Gene ATG12 in Potato Dry Rot Fungus Fusarium oxysporum.

Autophagy is an intracellular process in all eukaryotes which is responsible for the degradation of cytoplasmic constituents, recycling of organelles, and recycling of proteins. It is an important cellular process responsible for the effective virulence of several pathogenic plant fungal strains, having critical impacts on important crop plants including potatoes. However, the detailed physiological mechanisms of autophagy involved in the infection biology of soil-borne pathogens in the potato crop needs to be investigated further. In this study, the autophagy-related gene, FoATG12, in potato dry rot fungus Fusarium oxysporum was investigated by means of target gene replacement and overexpression. The deletion mutant ∆FoATG12 showed reduction in conidial formation and exhibited impaired aerial hyphae. The FoATG12 affected the expression of genes involved in pathogenicity and vegetative growth, as well as on morphology features of the colony under stressors. It was found that the disease symptoms were delayed upon being inoculated by the deletion mutant of FoATG12 compared to the wild-type (WT) and overexpression (OE), while the deletion mutant showed the disease symptoms on tomato plants. The results confirmed the significant role of the autophagy-related ATG12 gene in the production of aerial hyphae and the effective virulence of F. oxysporum in the potato crop. The current findings provid an enhanced gene-level understanding of the autophagy-related virulence of F. oxysporum, which could be helpful in pathogen control research and could have vital impacts on the potato crop.

TOR Inhibitors Synergistically Suppress the Growth and Development of Phytophthora infestans, a Highly Destructive Pathogenic Oomycete.

Phytophthora infestans, one of most famous pathogenic oomycetes, triggered the Great Irish Famine from 1845 to 1852. The target of rapamycin (TOR) is well known as a key gene in eukaryotes that controls cell growth, survival and development. However, it is unclear about its function in controlling the mycelial growth, sporulation capacity, spore germination and virulence of Phytophthora infestans. In this study, key components of the TOR signaling pathway are analyzed in detail. TOR inhibitors, including rapamycin (RAP), AZD8055 (AZD), KU-0063794 (KU), and Torin1, inhibit the mycelial growth, sporulation capacity, spore germination, and virulence of Phytophthora infestans with AZD showing the best inhibitory effects on Phytophthora infestans. Importantly, compared with a combination of RAP + KU or RAP + Torin1, the co-application of RAP and AZD show the best synergistic inhibitory effects on P. infestans, resulting in the reduced dosage and increased efficacy of drugs. Transcriptome analysis supports the synergistic effects of the combination of RAP and AZD on gene expression, functions and pathways related to the TOR signaling pathway. Thus, TOR is an important target for controlling Phytophthora infestans, and synergism based on the application of TOR inhibitors exhibit the potential for controlling the growth of Phytophthora infestans.

Ethylicin Prevents Potato Late Blight by Disrupting Protein Biosynthesis of Phytophthora infestans.

Phytophthora infestans, the causal agent of potato late blight, triggered the devastating Great Irish Famine that lasted from 1845 to 1852. Today, it is still the greatest threat to the potato yield. Ethylicin is a broad-spectrum biomimetic-fungicide. However, its application in the control of Phytophthora infestans is still unknown. In this study, we investigated the effects of ethylicin on Phytophthora infestans. We found that ethylicin inhibited the mycelial growth, sporulation capacity, spore germination and virulence of Phytophthora infestans. Furthermore, the integrated analysis of proteomics and metabolomics indicates that ethylicin may inhibit peptide or protein biosynthesis by suppressing both the ribosomal function and amino acid metabolism, causing an inhibitory effect on Phytophthora infestans. These observations indicate that ethylicin may be an anti-oomycete agent that can be used to control Phytophthora infestans.

Autophagy Related Gene (ATG3) is a Key Regulator for Cell Growth, Development, and Virulence of Fusarium oxysporum.

Fusarium oxysporum is the most important pathogen of potatoes which causes post-harvest destructive losses and deteriorates the market value of potato tubers worldwide. Here, F. oxysporum was used as a host pathogen model system and it was revealed that autophagy plays a vital role as a regulator in the morphology, cellular growth, development, as well as the pathogenicity of F. oxysporum. Previous studies based upon identification of the gene responsible for encoding the autophagy pathway components from F. oxysporum have shown putative orthologs of 16 core autophagy related-ATG genes of yeast in the genome database which were autophagy-related and comprised of ubiquitin-like protein atg3. This study elucidates the molecular mechanism of the autophagy-related gene Foatg3 in F. oxysporum. A deletion (∆) mutants of F. oxysporum (Foatg3∆) was generated to evaluate nuclear dynamics. As compared to wild type and Foatg3 overexpression (OE) strains, Foatg3∆ strains failed to show positive MDC (monodansylcadaverine) staining which revealed that Foatg3 is compulsory for autophagy in F. oxysporum. A significant reduction in conidiation and hyphal growth was shown by the Foatg3∆ strains resulting in loss of virulence on potato tubers. The hyphae of Foatg3∆ mutants contained two or more nuclei within one hyphal compartment while wild type hyphae were composed of uninucleate hyphal compartments. Our findings reveal that the vital significance of Foatg3 as a key target in controlling the dry rot disease in root crops and potato tubers at the postharvest stage has immense potential of disease control and yield enhancement.

Role of Autophagy-Related Gene atg22 in Developmental Process and Virulence of Fusarium oxysporum.

Autophagy is a universal catabolic process preserved in eukaryotes from yeast to plants and mammals. The main purpose of autophagy is to degrade cytoplasmic materials within the lysosome/vacuole lumen and generate an internal nutrient pool that is recycled back to the cytosol during nutrient stress. Here, Fusarium oxysporum was utilized as a model organism, and we found that autophagy assumes an imperative job in affecting the morphology, development, improvement and pathogenicity of F. oxysporum. The search of autophagy pathway components from the F. oxysporum genome database recognized putative orthologs of 16 core autophagy-related (ATG) genes of yeast, which additionally incorporate the ubiquitin-like protein atg22. Present study elucidates the unreported role of Foatg22 in formation of autophagosomes. The deletion mutant of Foatg22 did not demonstrate positive monodansylcadaverine (MDC) staining, which exposed that Foatg22 is required for autophagy in F. oxysporum. Moreover, the ∆Foatg22 strains exhibited a decrease in hyphal development and conidiation, and reduction in pathogenicity on potato tubers and leaves of potato plant. The hyphae of ∆Foatg22 mutants were less dense when contrasted with wild-type (WT) and overexpression (OE) mutants. Our perceptions demonstrated that Foatg22 might be a key regulator for the control of dry rot disease in tuber and root crops during postharvest stage.

Synergistic anti-oomycete effect of melatonin with a biofungicide against oomycetic black shank disease.

Human health, food safety, and agriculture have been threatened by oomycetic diseases caused by notorious pathogenic oomycetes. Chemical oomyceticides are the main approaches in control of pathogenic oomycetes. However, the overused chemical oomyceticides have resulted in serious environmental pollution and drug resistance. The eco-friendly bio-oomyceticides are required for sustainable development through screening synergistic drug combinations. In this study, Phytophthora nicotianae (P. nicotianae), as one of the most destructive oomycetic diseases in agriculture, was used as a model system to screen the novel bio-oomyceticides based on drug combination. The results showed that treatment of melatonin or ethylicin (IUPAC Name: 1-ethylsulfonylsulfanylethane) alone displayed similar phenotypes such as the inhibition of the hyphal growth, reduction of the cell viability, and suppression of the virulence of P. nicotianae. Importantly, melatonin and ethylicin shared the same targets of interfering with the amino acid metabolism, overexpressing apoptosis-inducing factor, and dysregulating the virulence-related genes. Furthermore, strong synergism against P. nicotianae was induced by combining melatonin with ethylicin. Under treatment of the combination of melatonin and ethylicin, the expression of genes associated with amino acid, the apoptosis-inducing factor, and the virulence-related genes was much more significantly dysregulated than that of single drug treatment. Thus, the tobacco black shank caused by P. nicotianae can be successfully controlled using the combination of melatonin and ethylicin. These observations suggest that the synergistic effect based on the combination of melatonin and ethylicin is an eco-friendly alternative for the control of the destructive oomycetic diseases.