A reference transcriptome for walnut anthracnose pathogen, Ophiognomonia leptostyla, guides the discovery of candidate virulence genes

Abstract

Despite the economic losses due to the walnut anthracnose, Ophiognomonia leptostyla is an orphan fungus with respect to genomic resources. In the present study, the transcriptome of O. leptostyla was assembled for the first time. RNA sequencing was conducted for the fungal mycelia grown in a liquid media, and the inoculated leaf samples of walnut with the fungal conidia sampled at 48, 96 and 144 h post inoculation (hpi). The completeness, correctness, and contiguity of the de novo transcriptome assemblies generated with Trinity, Oases, SOAPdenovo-Trans and Bridger were compared to identify a single superior reference assembly. In most of the assessment criteria including N50, Transrate score, number of ORFs with known description in gene bank, the percentage of reads mapped back to the transcript (RMBT), BUSCO score, Swiss-Prot coverage bin and RESM-EVAL score, the Bridger assembly was the superior and thus used as a reference for profiling the O. leptostyla transcriptome in liquid media vs. during walnut infection. The k-means clustering of transcripts resulted in four distinct transcription patterns across the three sampling time points. Most of the detected CAZy transcripts had elevated transcription at 96 hpi that is hypothetically concurrent with the start of intracellular growth. The in-silico analysis revealed 103 candidate effectors of which six were members of Necrosis and Ethylene Inducing Like Protein (NLP) gene family belonging to three distinct k-means clusters. This study provided a complex and temporal pattern of the CAZys and candidate effectors transcription during six days post O. leptostyla inoculation on walnut leaves, introducing a list of candidate virulence genes for validation in future studies.