First fine mapping of a strain of Rhizoctonia Solani AG-3, causing tobacco target spot

Abstract

Objectives

Rhizoctonia solani AG-3 is the casual pathogen of tobacco target spot, a serious fungal disease of tobacco that severely decreases yield and quality. To examine the pathogenic mechanisms of this fungus, it is crucial to understand its genetics. The objective of this work was to generate the first fine mapping of a R. solani AG-3 strain from tobacco and to explore potential virulence genes, which will lay the foundation for genetic characterization and its interaction with tobacco. The functional genes involved in this study can be used as the candidates for follow-up experimental analyses.

Data description

Rhizoctonia solani AG-3 strain XEMS25-1 was isolated from disease leaves of tobacco target spot in Enshi, Hubei Province, China. The DNA was sequenced using Pacific Biosciences Sequel II (PacBio) and Illumina NovaSeq PE150 (Nova). Data from both sequencing platforms were combined, and the de novo assembly yielded an estimated 39.4 Mb genome. Completeness of the genome examined using Benchmarking Universal SingleCopy Orthologs (BUSCO) showed that the assembly had 93.7% of the 758 genes in fungi_odb10. PHI (Pathogen Host Interactions) database analysis revealed 519 reduced virulence genes, 91 loss of pathogenicity genes, 28 hypervirulence genes and 18 effectors might be the pathogenicity-related genes in R. solani AG-3 strain XEMS25-1. These genes could be selected as the RNA-silencing targets for exploring the molecular mechanisms of R. solani AG-3 pathogenicity on tobacco.

Objectives

Tobacco is an important economic crop cultivated worldwide. In recent years, tobacco target spot disease, caused by the fungal pathogen Rhizoctonia solani AG-3, has emerged as a severe foliar disease [1, 2], widely affecting tobacco-growing regions in China [3,4,5,6]. The pathogen primarily overwinters in the soil and plant debris in the form of sclerotia or mycelia, serving as the main source of infection [7]. However, the molecular pathogenesis of R. solani AG-3 on tobacco is not thoroughly understood, nor the genomics of the pathogen.

A R. solani AG-3 strain, XEMS 25 − 1, was isolated from tobacco leaves in Enshi, Hubei Province, China in 2022. This strain was identified as the causal agent of tobacco target spot and verified as R. solani AG-3 fusion group [5]. To date, the draft genome of R. solani AG-3 from potato has been sequenced based on second generation sequencing [8], but an isolate from tobacco has not been reported and fine mapping of R. solani AG-3 strain also has not been generated. In this study, whole genome sequencing using hybrid second and third generation sequencing techniques on R. solani AG-3 was performed, and the sequences assembled. This study provides a reference for detailed genome-wide mapping and genome annotation of R. solani AG-3 for researchers. It also provides a theoretical basis for screening the pathogenic genes and related pathways of R. solani AG-3, and clarifying the pathogenic mechanisms of tobacco target spot disease.

Data description

The tobacco target spot strain, XEMS 25 − 1, collected from Enshi, China (29°07′10″N, 108°23′12″E) was used for genomic DNA extraction. Isolation, pathogenicity test and identification of the strain XEMS 25 − 1 were performed based on previous work [5]. Hyphae were transferred onto potato dextrose agar and cultured at 25 °C for 72 h. Hyphae were collected, and DNA was extracted using NEBNext^®Ultra™ DNA Library Prep Kitfor Illumina (NEB, USA) kit. A sample of 2 µg/ml DNA was sequenced specifying ~ 350 bp fragments on an Illumina PE150, to obtain 6,049 Mb of raw data (Data file 1, Data set 1, Table 1) [9, 10]. After filtering the raw data, there were 5,609 Mb of clean data. An analysis based on K-mer statistics was used to estimate genome size of 48.14 Mb before genome assembly (Data file 2, Data set 1, Table 1) [9, 10]. Illumina data were used for initial assessment and correction of the genome. The genomic DNA of XEMS 25 − 1 was then extracted using an in-house method called GT1 [11], after which the purity and integrity of the DNA was measured by agarose gel electrophoresis and quantified using Qubit. A library was constructed using the SMRT bell TM Template kit (version 2.0) [12], and sequenced using the PacBio platform. This generated 12.05 Gb of data with 738,951 subreads, and average subread length of 16,301 bp (N50 = 18,183 bp, N90 = 13,636 bp) (Data file 3, Data set 1, Table 1) [9, 10]. Based on the clean data of the third-generation sequencing data after quality control of each sample, the reads were assembled using Falcon software (https://github.com/PacificBiosciences/FALCON/) [13], and then Racon (version: 1.4.13) [14] software for three rounds of error correction based on the third-generation sequencing data, and then three rounds of Pilon (version: 1.22) [15] error correction with second-generation read data to obtain the final assembly results. The cumulative assembly resulted in 26 contigs, with an N50 of 2,548,437 bp (Data file 4, Data set 2, Table 1) [10, 16]. The completedness of the assembled genome was assessed using BUSCO software [14], and the genome was found to contain 710 of the 758 BUSCO genes (93.7%), with 705 complete single copies (93%), 5 duplicates (0.7%), and 5 fragmented (0.7%), with 42 BUSCO genes missing (5.6%) (Data file 5, Table 1) [10, 17]. The XEMS 25 − 1 genome was then annotated and genome-wide mapped against R. solani AG-3 Rhs1AP as reference [8]. The predicted number of genes was 10,317 using De-novo Augustus prediction (Data file 6, Data set 1, Table 1) [9, 10, 18]. There were 3,367 tandem repeats in the assembly (Data file 7, Table 1) [10, 19]. SignalP (Version 4.1) [20] and TMHMM (Version 2.0c) were used for detection of signal peptides and transmembrane structures, respectively, identifying 741 secreted proteins (Data file 8, 9, 10, Table 1) [10]. Genome basic annotation was performed by alignment and functional annotation against five major databases (NR [21], Swiss-Prot [22], Pfam, GO, and KEGG) [23]. In the gene function analysis, 10,076 genes were annotated by comparison with the NR database; 2,485 with the Swiss-Prot database; 6,049 with the PFAM database; 6,049 by GO database annotation; and 6,005 by alignment annotation with the KEGG database (Data file 8, Table 1) [10]. The assembled genome sequence for Rhizoctonia solani AG-3 strain XEMS 25 − 1, combined with the prediction results of coding genes, were depicted using a visualization tool Circos software (Table 1) [10, 24]. PHI database was then used to predict potential pathogenicity genes and a total of 983 PHI-related genes were identified in the R. solani XEMS25-1 strain, including 519 reduced virulence genes, 91 loss of pathogenicity genes, 28 hypervirulence genes, 18 effectors and others (Data file 11, Table 1) [10]. These candidate genes might be the RNA-silencing targets using Host-induced Gene Silencing technology for exploring the molecular mechanisms of R. solani AG-3 pathogenicity on tobacco. The XEMS25-1 genome was used to compare with the potato Phs1AP genome, and the results revealed that the XEMS25-1 genome contains 5,306 core genes and 3,330 specific genes, indicating genome variation might be exist between R. solani AG-3 strains from different host. The clean data of the XEMS25-1 genome were deposited in NCBI with Bioproject PRJNA1193463 and Biosample SAMN45140045 (Data set 1, Table 1) [9]. The assembly data were deposited in CNCB with Bioproject PRJCA034663 and Biosample SAMC4533655 [16]. Other results files are available on the figshare website https://doiorg.publicaciones.saludcastillayleon.es/10.6084/m9.figshare.27960000.v4 [10].

Table 1 Overview of all data files/data sets

Limitations

The data generated in this study is limited to a single genome sequence. We can not get more information of host specificity or adaptation of the pathogen. To overcome this limitation, other strains of tobacco target spot fungus R. solani AG-3 from different locations should be sequenced. Particularly, R. solani AG-3 strains with different pathogenic characteristics could be used for whole genome sequencing. Furthermore, transcriptome data of key infection stages of R. solani AG-3 on tobacco would be helpful to elucidate the pathogenic mechanism.