Alternaria radicina xylanase is required for the occurrence of carrot black rot disease

Alternaria radicina, the causative agent of carrot black rot, causes significant damage to both the leaves and roots of carrots throughout the growing season. The pathogenic effectors are seldomly isolated and lack of functional studies. In this study, we performed genomic sequencing, assembly, and annotation of an A. radicina isolate. A. radicina genome is about 34.71 Mb, and contains 11,271 coding sequences. Based on the Pathogen Host Interactions (PHI) database, three xylanase genes containing the glycoside hydrolase 11 (GH11) domain are enriched. We further identify five xylanase genes (ArXyn1 ∼ ArXyn5) in A. radicina genome according to the amino acid sequence similarity, and investigate their functional roles in the infection process. The transcriptions of ArXyn1 and ArXyn3 are significantly induced in both A. radicina strain and infected leaf tissues during the infection. Transiently expressing ArXyn1 or ArXyn3 in Nicotiana benthamiana leaves triggers the cell death, which is dependent on the signal peptide localized at the N-terminal of each gene. Subcellular localization analysis reveals that both ArXyn1 and ArXyn3 are localized in the plasma membrane, cytosol, nucleus and extracellular spaces of N. benthamiana leaf cells. Deleting either ArXyn1 or ArXyn3 slightly reduces the fungal growth on the medium. The ArXyn1 or ArXyn3 deletion mutant cause less severe disease symptoms on carrot leaves and roots than wild type strain. Scanning electron microscopy reveals that both mutants cause reduced cell wall damages compared to wild type strain. Taken together, our results suggest that xylanases contribute to A. radicina virulence, and play important roles in the occurrence of carrot black rot disease.