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Neopestalotiopsis cubana Maharachch. is the primary pathogen responsible for leaf spot and fruit ring rot in guava (Psidium guajava L.). The ADP-ribosylation factor-like protein 8 (Arl8) belonging to the ADP-ribosylation factor (Arf) superfamily is associated with lysosomal processes including localization, trafficking, and fusion. The function of Arl8 in phytopathogenic fungi remains poorly understood. Objective To elucidate the function of Arl8 in N. cubana. Methods An Arl8 gene was identified from the genome of N. cubana and designated as NecArl8. A NecArl8 knockout mutant (ΔNecArl8) and a complemented strain (ΔNecArl8-C) were generated via clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9, (CRISPR/Cas9)-mediated genome editing. These strains were subsequently analyzed through phenotypic experiments and RNA-seq. Results ΔNecArl8 showed a reduction of 34.93% in sporulation capacity and significant increases of 5.74% and 7.39% in inhibition rates by salt and osmotic stresses, respectively. In addition, this mutant exhibited weakened pathogenicity, as indicated by a reduction of 5.74 mm in the average spot diameter. In contrast, the mutant displayed significantly enhanced resistance to oxidative stress, which showed an inhibition rate of -19.22%. After long-term culture, a large number of vacuolated cells were observed in the mutant. RNA-seq analysis was performed on ΔNecArl8 and the wild-type strain to identify differentially expressed genes. Transcriptomic analysis showed that 2 330 genes were significantly down-regulated and 1 355 genes were significantly up-regulated. Gene set enrichment analysis further revealed that transmembrane transport proteins were broadly suppressed, and the expression levels of 16 fungal-type vacuolar-related proteins were significantly down-regulated, suggesting that the structure and functions of fungal-type vacuoles were seriously damaged. The reverse transcription quantitative PCR (RT-qPCR) results showed the expression patterns of 9 randomly selected transmembrane transport genes were consistent with those obtained by transcriptome sequencing, indicating that the transcriptome data were highly reliable. Conclusion NecArl8 may affect the pathogenicity of N. cubana on guava by regulating function of transmembrane transport. This study lays a theoretical foundation for exploring the pathogenic mechanism of N. cubana and developing new control strategies.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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