The G. citri-aurantii genome sequencing, assembly and annotation.
The genome of G. citri-aurantii was sequenced by PacBio RS II. A 20-Kb library was constructed for genomic DNA. After filtering the original data, a total of 2.54 Gb of high-quality sequencing data was obtained (Fig 1A). Assembly led to a genome size of 28.10 Mb (28,101,572bp) with approximately 90x coverage, divided into 29 scaffolds with a minimum size of 1 Mb, as well as an overall G + C content of 38.6% (Table 1). And an overall repeat content of 17.93% of the genome was discovered through RepeatMasker analysis and PASTEClassifier classification. Some characteristics of G. citri-aurantii genome are shown in Table 1.
6,783 genes were predicted by BRAKER software, with an average length of 2,058 bp, a gene density of 241 genes/Mb and a total of 46.8% of the genome covered by protein-coding genes. There is an average exon and intron length of 1,203bp and 111bp.
Ten chromosome groups can be clearly distinguished according to the evaluation of HI-C assembly results (Fig. 1C) and displayed by circus plot (Fig. 1D). Within each group, the intensity of the interaction at the diagonal position is higher than the position of the nondiagonal line, and the results indicated that the interaction between adjacent sequences (diagonal positions) is higher in the Hi-C assembled chromosome but not in nonadjacent sequences (nondiagonal position), which is consistent with the principle of Hi-C helper genome assembly. After Hi-C assembly, the genome sequence length of 27.94 Mb with 193x coverage was mapped to the chromosome, accounting for 99.43% of the previously measured G. citri-aurantii genome. In the sequence mapped to the chromosome, the sequence length and direction can be determined to be 27.58 Mb, accounting for 98.63% of the total length of the sequence located on the chromosome (Table 1).
In the genome structure, TEs and repetitive DNA sequences play important roles in the evolution of fungi14. The total repeat sequences of 5,039,960 bp (5 M) were identified, accounting for 17.93% of the G. citri-aurantii total genome, including LTR retrotransposon, DNA transposon, transposon tandem and other unclassified repeat sequences (Fig. 2A). The number of repeats reached 11,476. Interestingly, Rest of genome are the most repeating sequence (82.17%). Notably, LINE and LTR account for 5.1 and 2.31%, respectively.
Recently, DNA methylation has also been reported in the class Dothideomycetes15. In G. citri-aurantii genome, 171,537 m4C (4-methyl-cytosine) and 11,765 m6A (6-methyl-adenosine) were identified (Fig. 2B). Most of the categorized DNA methylations are m4C. However, m6A DNA methylations occur with high frequency in the regions of repetitive elements compared with m4C (Fig. 2C).
Using the predicted protein sequence of the gene to perform BLAST alignment with functional databases, such as the TCDB database and the PHI database, 170 TCDB and 1,933 PHI were predicted. Then, 196 CAZyme and 1,270 Transmembrane proteins were found through analysis of protein sequences of all predicted genes (Fig. 2D). In G.citri-aurantii genome, The protein containing the transmembrane helix was removed from the predicted 339 proteins containing the signal peptide, and the remaining 158 proteins were secreted proteins. Through domain calling analysis, PF14295.4 (n=6) is the most abundant domain, which mediates protein/protein interactions.
Comparative genomic analysis
The evolutionary relationship of G. citri-aurantii and other fungi species was analyzed using a group of phylogenetic backbone genes of the fungi. Phylogenetic analysis revealed that G. citri-aurantii is evolutionarily close to Galactomyces citri-aurantii, a plant pathogen that has been found on citrus fruit or in the soil of citric fruit orchards (Fig. 3A). In addition, G. citri-aurantii is also close to the other two fungi of Dipodascaceae sp. and Geotrichum candidum (Fig. 3A). To date, in all fungi in the phylogenetic tree of G. citri-aurantii, only the genome of Geotrichum candidum has been published. Synteny analysis of the G. citri-aurantii genome and G. candidum genome revealed that the G. citri-aurantii genome displays different synteny than those fungi (Fig. 3B,C).
Transcriptome analysis on the guazatine-resistance of G. citri-aurantii.
The guazatine-resistance transcriptome data was repeatable between samples (Fig. 5C). There were 714 DE genes (FDR=2 and FC = 0.05) between AY-1 and AY1-68 of G. citri-aurantii before guazatine treatment and were used as cutoff values (Fig.5E). Among these genes, 385 genes were upregulated, and 329 genes were downregulated. The results indicated that the gene expression pattern was changed significantly in the resistant strains. After guazatine treatment, there were 226 DE genes in AY1-68 that contained 52 downregulated and 174 upregulated DE genes. In addition, 226 DE genes belonging to AY1-68 were uniquely associated with the resistance of AY1-68 to guazatine. There were 108 DE genes between G1 and G3 after guazatine treatment, and they were associated with the resistance of AY1-68 to the drug response of guazatine.
All 1146 DE genes and selected genes were classified by Gene ontology (GO) consortium analysis (Fig. 5F). Compared with the wild strain AY-1, the drug-resistant strain AY1-68 could increase its resistance several hundredfold. It is suspected that some of the cellular component organization of this strain has changed to some extent. Simultaneous sequencing revealed that 80% of these genes have increased expression levels of the transcriptome data. These results revealed that the cellular component of the drug-resistant strain AY1-68 changed.
To identify genes related to the responses of G. citri-aurantii to the antifungal drug guazatine and the high resistance of G. citri-aurantii, differentially expressed genes of the ABC transporter family and MFS transporter family were further analyzed. The two genes (EVM0003677, EVM0005437) of the ABC transporter family and six transporter genes (EVM0000087, EVM0000766, EVM0001093, EVM0003101, EVM0004095, EVM0005235) of the MFS transporter family received more attention. In this study, RT-qPCR was used to validate the expression levels of eight transporter genes (Fig. 6A, C). these results indicate that overexpression of these genes may enhance the drug resistance of G. citri-aurantii.
Transcriptome data analysis revealed that seven MATE transporters (EVM0000063, EVM0002368, EVM0001080, EVM0002368, EVM0003579, EVM0005976, EVM6710) were related to drug resistance, and these transporter genes were not reported before this study. In the guazatine-resistant strain AY1-68, the expression levels of two MATE genes (EVM0000063, EVM0002368) were upregulated compared with the guazatine-susceptible strain AY-1 (Fig. 6B). The other five MATE genes had decreased expression. Notably, these MATE transporters were normal in AY1-68 after guazatine treatment. These results indicate that two ABC genes and six MFS genes of G. citri-aurantii were activated after guazatine treatment. However, two MATE genes may play a decisive role in enhancing drug resistance.