Identification of UvAtg8
To identify the Atg8 homologous protein in U. virens, the amino acid sequence of Atg8 (GenBank KZV12988) from Saccharomyces cerevisiae was used as a query for the BLASTP search on NCBI. The KDB12146.1 (termed as UvAtg8) was hit as the ortholog of S. cerevisiae Atg8 with 78% identity and 90% similarity (Fig. 1a). UvAtg8 consists of 121 amino acids. Sequence analysis using motif scan (https://myhits.isb-sib.ch/cgi-bin/motif_scan) revealed that UvAtg8 contained a MAP 1_LC3 domain between 13 and 116 amino acids. The phylogenetic analysis results of the amino acid sequences of Atg8 from U. virens, F. graminearum, Aspergillus niger, Neurospora crassa, Metarhizium brunneum, Sclerotinia sclerotiorum, M. oryzae, Coprinopsis cinerea, and S. cerevisiae, revealed that UvAtg8 protein was most similar to Atg8 of M. brunneum and S. sclerotiorum. These results indicated that Atg8 is highly conserved in different species, and also suggested that UvAtg8 may have important functions in U. virens (Fig. 1b).
Disruption and Complementation of UvATG8
To understand the biological functions of UvATG8, UvATG8 deletion mutants were generated by targeted gene replacement in the wild type (WT) HWD-2 strain (Fig. 2a). Southern blot assay was performed to confirm the targeted gene replacement events and excluding ectopic integrations. The appearance of the 3.8 Kb (kilobases) band in the ∆Uvatg8 mutant, with concomitant loss of the WT 2.4 Kb in UvATG8 locus, indicated that the gene replacement event was correct (Fig. 2b). The resultant three ∆Uvatg8 mutants showed comparable phenotypes and formed smaller colonies than the WT, and ∆Uvatg8–36 and 102 were chosen for further experiments.
To confirm that the phenotypic differences observed in the ∆Uvatg8 mutants were all associated with the gene replacement event, a vector pFGL820-UvATG8 containing a full-length gene copy of UvATG8 with its native promoter was transformed into ∆Uvatg8–36. The resultant ∆Uvatg8-C strain was further confirmed using qRT-PCR (quantitative Real-time PCR) assay. The expression levels of UvATG8 in the WT and ∆Uvatg8-C strain were comparable, which indicated that ∆Uvatg8-C had rescued the expression of UvATG8 (Fig. 2c). Moreover, the ∆Uvatg8-C strain was similar to the WT strain in colony morphology, suggesting that ∆Uvatg8-C functionally complemented the phenotype of ∆Uvatg8.
UvAtg8 Could Sever as a Marker for Autophagy in U. virens
Atg8 is known to be one of the key components of autophagy (Liu et al. 2016; Zong et al. 2016; Hofius et al. 2017). To determine whether UvATG8 is required for autophagy in U. virens, the changes in the process of autophagy in the WT and ∆Uvatg8-C strains were determined using microscopy (Fig. 3a). When cultured in SD-N (synthetic dropout medium without nitrogen) liquid medium in the presence of 3 mM PMSF (Phenylmethanesulfonyl fluoride) for 4 h, less than 10% of the vacuoles had a very few autophagic bodies in the ∆Uvatg8 mutant. In contrast, nearly 65% of the vacuoles in the WT strain exhibited multiple autophagic bodies. These results suggested that the autophagic pathway was blocked in the ∆Uvatg8 mutant.
To observe the localization of UvAtg8, the U. virens strain expressing GFP-UvAtg8 fusion protein under native regulation and as the sole copy of UvATG8 was generated. The size of GFP-UvAtg8 protein was correct and the morphologies of the GFP-UvATG8 strain were comparable to the WT strain, indicating that the GFP-UvAtg8 fusion protein was functional in U. virens. In order to induce autophagy, the GFP-UvATG8 strain was cultured in a liquid PS (Potato sucrose) medium for 2 d, and then inoculated into the SD-N medium in the presence of 3 mM PMSF. During the growth process in the PS medium, the GFP signals were weak and in punctate structures in the GFP-UvATG8 strain. However, following the growth in SD-N medium for 12 h, the GFP fluorescent signals increased and could be easily observed in the CMAC (7-amino-4-chloromethylcoumarin) stained vacuoles (Fig. 3c, d). These results indicated that the GFP-UvAtg8 was localized in the cytoplasm as pre-autophagosomal structures under general conditions, but were predominantly accumulated in the lumen of the vacuoles under nitrogen starvation condition.
Next, the Western blot assay was performed to analyze the expression of GFP-UvAtg8 with anti-GFP antibody under both normal and nitrogen starvation conditions. Based on the predicted sizes and relative mobility, the two detected bands were judged to be the full-length GFP-UvAtg8 and likely the free GFP (Fig. 3b). The stronger GFP band indicated enhanced expression and degradation of the GFP-UvAtg8 in the SD-N medium, and indirectly showed enhanced autophagic response in U. virens under nitrogen starvation condition.
Taken together, these results suggested that UvAtg8 is essential for autophagy in U. virens, and GFP-UvAtg8 could serve as an appropriate marker for monitoring autophagy in U. virens as GFP/RFP-Atg8 in other organisms (Deng et al. 2009; Zong et al. 2016).
UvAtg8 Plays Important Roles in Vegetative Growth
Because the ∆Uvatg8 mutants had formed smaller colonies than the WT strain, the vegetative growth of the WT, ∆Uvatg8, and ∆Uvatg8-C strains were determined using different mediums. The ∆Uvatg8 strain was slightly reduced in mycelia growth when compared with that of the WT strain on the PSA. However, the mycelia growths of the ∆Uvatg8 strain on the SD and SD-N medium were significantly reduced (Fig. 4a,b). Notably, the inhibition rate of the ∆Uvatg8 strain was significantly increased when compared with that of the WT strain under the nitrogen starvation condition, which indicated that its defects in hypha growth were partially dependent on the nutrient conditions (Fig. 4a,c). In contrast, the mycelia growth and colony morphology were rescued in the ∆Uvatg8-C strain (Fig. 4), suggesting that UvAtg8 plays important roles in the vegetative growth of U. virens.
UvAtg8 Is Involved in Various Stress Responses
Autophagy is known to be related to several stress responses (Yin et al. 2019). However, it remains unclear whether UvATG8 regulates stress responses of U. virens. Therefore, the sizes of ∆Uvatg8 colonies were measured under different stress conditions, including oxidative, hyperosmotic, and cell wall stresses. In the presence of 0.03% H2O2, the growth was reduced by 50% in the WT strain, but approximately 75% in the ∆Uvatg8 mutant after incubation for 15 d (Fig. 5). These results indicated that the ∆Uvatg8 was sensitive to oxidative stress. Similarly, both the ∆Uvatg8–36 and 102 had shown significantly increased inhibitory effects when compared with the WT strain on the medium with 0.4 M NaCl or 0.7 M Sorbitol (Fig. 5), thereby suggesting that UvATG8 contributed to the adaptions to the hyperosmotic stresses. In addition, the deletion of UvATG8 resulted in decreased tolerance to different cell wall stresses (Fig. 5), including 0.03% Sodium dodecyl sulfate (SDS), 200 μg/mL Calcofluor white (CFW), and 240 μg/mL Congo Red (CR), in U. virens. The complementation strain ∆Uvatg8-C showed similar phenotypes as the WT strain under all of the observed stress conditions. All the aforementioned results indicated that UvAtg8 is involved in the adaptions to oxidative, hyperosmotic, and cell wall stresses in U. virens.
UvAtg8 Is Required for Pathogenesis in U. virens
To investigate the roles of UvATG8 in the virulence of U. virens, infection assays were performed. The WT, ∆Uvatg8–36 and 102, and ∆Uvatg8-C strains were inoculated into panicles of the susceptible rice cultivar Wanxian 98. The formation of rice false smut balls were calculated 3 weeks after the inoculations. Remarkably, the numbers of smut balls in the ∆Uvatg8–36 and 102 strains were significantly lower than those in the WT and complementation strains (Fig. 6a). Therefore, deletion of UvATG8 significantly attenuated U. virens virulence, suggesting that UvATG8 is a key regulator of the pathogenicity of U. virens.
U. virens not only occupies rice grains, but also produces compounds, eg. Ustilaginoidins O, E, F and isochaetochromin B2, which are toxic to rice seeds (Lu et al. 2015). In order to determine the inhibitory effect of toxic compounds on the germination of rice seeds, the filtrates were isolated from the PS cultures of 7 d old WT, ∆Uvatg8–36 and 102, and ∆Uvatg8-C strains to treat rice seeds. As shown in Fig. 6d, the shoots of the rice treated by the filtrate of the ∆Uvatg8 strain culture was significantly longer than those of the WT and ∆Uvatg8-C strains. Furthermore, the expression level of UvUSTA, which is a member of the gene cluster responsible for ustiloxin synthesis, in the deletion mutant of UvATG8 was lower than that of the WT strain (Fig. 6e) (Tsukui et al. 2015; Zheng et al. 2016). These findings suggested that UvATG8 may have produced fewer toxic compounds which inhibit the shoot growth of rice shoots during rice seed germination.
Serious Virulence Defects in ∆UvAtg8 Were Mainly Caused by the Highly Decreased Formation of Secondary Spores
To further investigate the serious virulence defects caused by deletion of UvATG8, the infection processes of the WT, ∆Uvatg8, and ∆Uvatg8-C strains were determined. Although the structures of the conidia appeared to be normal, the conidial production of ∆Uvatg8 mutant was highly reduced in comparison with that of the WT and ∆Uvatg8-C strains (Fig. 7a,b). Whereas the WT strain had formed 4.8 × 106 conidia/mL in 7 d old cultures, the ∆Uvatg8 mutants had produced less than 0.1 × 106 conidia/mL under the same conditions. This may have been the reason for the virulence deficiency of the ∆Uvatg8 strain. Furthermore, the GFP-UvAtg8 was likely presented in the vacuoles during conidiation (Fig. 7c), indicating that autophagy had occurred at that stage. However, when the same concentrations of conidia were inoculated, the infection efficiency of the ∆Uvatg8 strain was still observed to be highly decreased (Fig. 6a,b). Therefore, the results showed that, with the exception of the slightly slower growth and lower conidiation in the ∆Uvatg8 strain, there were definitely other reasons for the virulence deficiency of the ∆Uvatg8 strain.
Next, the germination of the conidia from ∆Uvatg8 mutant was detected. There were no significant differences observed in the germination of conidia between the WT strain and ∆Uvatg8 mutant. However, it should be noted that the formation of secondary spores was highly reduced in the ∆Uvatg8 mutant on the rice surface (Fig. 7d). During pathogenic processes of U. virens, the formation of secondary spores tends to greatly increase the amount of inoculation available to infect rice plants (Fan et al., 2013). Therefore, the highly reduced formation of secondary spores may have been the main reasons for the pathogenicity defects in the ∆Uvatg8 mutant. Meanwhile, the expression of GFP-UvAtg8 was highly induced and targeted to the vacuoles (Fig. 7e, S1), suggesting that the UvAtg8 mediated autophagy occurrences and played an important role in formation of secondary spores during the infection of rice.