| Cylindrocladium black rot of peanut is caused by Calonectria ilicicola.The disease can damage peanut roots,stems,leaves,and fruits,and cause rot,and plant death,resulting in serious yield loss.In this research,whole genome sequencing of Cylindrocladium peanut black rot strain Calonectria ilicicola 14017(referred to as Ci14017)was performed.Through comparative genomics,the transcriptome and proteome of the interactions of pathogen with resistant and sensitive peanut lines were analyzed to identify 48 pathogenicity-related genes.Gene knockout of one of genes in the fungus was explored.The results are helpful for further mining and functional verification of the pathogenic genes of Calonectria ilicicola,and will be useful for studying pathogenesis.The main results and findings are as follows:1.Whole genome sequence analysis of Ci14017 was done.The highly pathogenic strain Ci14017 was selected for whole-genome sequencing and compared with the published whole-genome genomes of comparative species.Paired-end sequencing was performed using Illumina/Solexa high-throughput technology and assembled by AbySS,SOAPdenovo,and Velvet.The optimal assembled genome size was 66 Mb,GC content was 48%,and 18,366 predicted genes were obtained.After the quality test by BUSCO software,the matching rate with the fungi core gene database reached 99%,indicating that a high-quality genome and its predicted genes were obtained.From the whole genome sequence,11841,17639,10116,8937 and 8805 genes were annotated in the SwissProt,NR,GO,KOG and KEGG databases,respectively.Analysis of secreted pathogenic genes on genomic sequences.Among 1,718 secreted proteins,168,117,318,225,549,and 431 genes were obtained from the MERPOS,DFVF,Lipase,Cytochrome P450,PHI,and CAZymes database,respectively.Genomic colinearity analysis showed that 6052 genes in the Ci14017 had colinearity with 6044 genes in Fusarium graminearum.Eighteen similar species were selected for comparative genomics analysis.It was found that the relationship between Ci14017 and Calonectria canadiana cc13393 was the closest.The time of differentiation was estimated to be 20.18 million years ago.The number of gene families expanded by 1217,and contracted by 627.In comparison with five other closely related Sordariomycete fungal genomes,Ci14017 had 135 specific single-copy gene families not found in the other genomes.2.Ci14017 and peanut interaction transcriptome analysis.The black rot highlyresistant line T09 and highly-sensitive line P562 were tested.Transcriptomes of axenic cultures on PDA were used as controls.High-throughput RNA sequencing technology was used to analyze the different stages of interaction between Ci14017 and resistant/sensitive peanuts(0,3,5,7,9 dpi)for transcriptome analysis.The generated sequences were compared with Ci14017 genomic sequences to obtain a total of 10,311 genes with in planta expression.Among them,1739,397,720,and 2432 genes were respectively induced with expression in P562 on days 3,5,7,and 9 after Ci14017 inoculation.In contrast,281,1652,1814,and 2515 genes were respectively induced with expression in T09 on days 3,5,7,and 9 after Ci14017 inoculation.At the same time points,the two lines were compared,and the differentially expressed genes of P562/T09 inoculated with Ci14017 were 92,105,301 and 90,respectively,at 3,5,7 and 9 days after inoculation.The generated sequences were compared with the whole genome sequence of peanut,and the differentially expressed genes of P562 and T09 before and after Ci14017 inoculation were 15621 and 18122,respectively.The two lines had 73 genes up-regulated and 56 genes down-regulated.3.Proteome analysis of Ci14017 and peanut interactions.Using highly-resistant line T09 and highly-sensitive line P562 peanuts as materials,non-labeled quantitative differential proteomics technology(Label Free)was used to perform a proteomic analysis of peanut tissue inoculated 9 days with Ci14017.A total of 9706 proteins were identified,including 1859 from Ci14017 and 7847 from peanuts.Among the quantifiable 6157 peanut proteins from tissue inoculated 9 days,T09 had 785 upregulated genes and 310 down-regulated genes compared to P562.Among the quantifiable 805 Ci14017 proteins,536 were up-regulated and 0 were down-regulated in peanut line P562 compared to T09.4.Transcriptome and proteome analysis.Inoculated peanut lines P562 and T09 were compared.A total of 142 up-regulated genes were found both in transcriptome and proteomic.Among them,75,7,23,32,7,30 and 9 genes were found in the PHI,DFVF,CAZymes,Lipase,MEROPS,Cytochrome P450 and the secreted protein database,respectively,of which 43 were pathogenicity-related genes.Transcriptome and proteome analyses shared 15 genes that were expressed only in P562 after inoculation with Ci14017,of which 6 genes were associated with pathogenicity.GO and KEGG enrichment analysis was performed on the 154 fungal genes that showed up-regulated expression,and considering only those genes identified after inoculation with Ci14017 were deduplicated,these were mainly enriched in the Mitochondrial and Exosome groupings.48 pathogenicity-related genes were compared with the NR database,including ethanol oxidase,chitin synthase,and cytochrome P450 enzymes.The protein-protein interaction analysis of 48 pathogenicity-related genes were performed showing 47 of them had the interaction relationship.5.Exploration of Ci14017 gene knockout system.A secreted gene g10687.t1 of Ci14017 was selected for gene knockout test.The gene of g10687.t1 was cloned and the binary plasmid pFGL821 was used to construct a gene knockout vector.Identification by PCR and enzyme digestion confirmed that the gene knockout vector pFGL821-g10687.t1 was successfully constructed and successfully transformed into the agrobacterium AGL-1.The exploration of this system lays the foundation for further transformation of Ci14017 for genetic verification. |
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