| Banana wilt,caused by fungal pathogen Fusarium oxysporum f.sp.cubense?Foc?,is one of the most important constraints on banana production and cause serious economic losses worldwide.Foc race 1 wiped out the banana industry based on cultivar Gros Michel in the middle of the last century.This forced the trade to shift to resistant cultivars of Cavendish variety.However virulent strain of race 4,to which Cavendish is susceptible,was rapidly spreading to banana production areas and is considered a major threat to banana production.Understanding molecular basis of the difference in virulence between two races will facilitate effective control of the disease.In this study,RNA sequencing was performed to compare genome-wide transcriptional profiles between Foc1 and Foc4 cultures grown in media containing banana cell wall,pectate or glucose as sole carbon source.The main objective of this study is to get further insight into the Foc genes that participate in banana cell wall decomposition and contribute to pathogenicity of the pathogen to banana host.Base on the RNA-seq analysis,two constitutively expressed endopolygalacturonase gene was cloned from Foc1 and Foc4.The enzyme activity and biochemical characterzation of end-PG encoded by pgc7 of Foc were identified by heterologous expression in Pichia pastoris.Delection of pgc7 in Foc resulted in reduction reduction of PG expression but had no affect on virulence.The fulfilment of this study could facilitate us to a better comprehension of plant-Fusarium interactions and more effective methods for the management of the disease.The major results of this study were as follows:1.RNA sequencing and functional gene annotation of the Foc transcriptome.Six cDNA libraries prepared from the pooled total RNA extracted from two Foc isolates grown under three carbon conditions were paired-end sequenced on the Illumina HiSeq2000 platform.The clean reads were assembled into 38,950 transcripts with an average length of 1937 bp and an N50 length of 3235 bp.The longest transcript for each locus was defined as the unigene,resulting in 23,417 unigenes with an average length of 1419 bp and an N50 length of 2542 bp.The functional annotation of all 23,417 assembled unigenes was mainly based on BLAST homology searches against seven public protein databases?Nr,Nt,KO,SwissProt,PFAM,GO and KEGG?.A total of 20,879 unigenes were annotated in at least one database,accounting for 89.16%of the unigenes.Using GO database to further annotation,the unigenes related to fungal pathogenicity were categorized into the GO terms‘regulation of biological process',‘response to stimulus',‘signaling',‘immune system process',‘growth',‘extracellular region',‘nucleic acid binding transcription factor activity',‘transporter activity'and‘antioxidant activity',etc.According to the function of fungal pathogenicity gene,a much larger number of the unigenes encoding CAZyme,PHI,secreted protein,protein kinase,transcription factor,transporter and cytochrome P450,and unigenes involved in secondary metabolites biosynthesis and cell autophagy were analyzed using different databases.2.Differentially expressed genes of Foc induced by host cell wall polysaccharides.Having generated a Foc reference transcriptome,genes displaying significant changes in expression when Foc was grown with host cell wall polysaccharides were identified.And The great majority of Foc genes are differentially expressed in a variety-specific manner during decomposition of the host cell wall.The expression profiles of gene encoding CAZyme,transcription factor and transporter were significantly different in Foc1 and Foc4.Pectinases and hemicellulases may contribute greatly to the ability of Foc to decompose the host cell wall.Zinc finger,bZIP and Zn?II?2CyS6 transcription factors was found to be specifically activated in Foc4 during the degradation of host cell wall polysaccharides.The unigenes sorted to PK?ABC?APC?Myb and VIC transporter superfamily were expressed with high level in Foc4,whereas the unigenes in MFS superfamily were expressed with high level in Foc1.In addition,more pathogenesis-related genes encoding secreted protein,protein kinase and cytochrome P450 and unigenes involved in secondary metabolites biosynthesis and cell autophagy were specifically differentially expressed in Foc4 as compared to Foc1.Much more PHI genes were differentially expressed in Foc4 as compared to Foc1 during degradation of host cell wall.Meanwhile,many genes with high expression level were fuctional unkowen,which may be potential pathogenicity-related genes of Foc.3.Cloning and heterologous expression of constitutively expressed endopolygalacturonases genes of Foc.Two constitutively expressed polygalacturonase gene from Foc1 and Foc4 were cloned and identified.The recombinant PGC7s were sucessfully expressed in Pichia pastoris as active extracellular endo-polygalacturonases.The Km and Vmax values of recominant PGC7from FOC4 were 0.89 mg·mL-1 and 99.01 Units·mg-1·min-1,respectively;while the Km and Vmax values of recominant PGC7 from FOC1 were 0.85 mg·mL-1 and 98.04Units·mg-1·min-1,respectively.Both recombinant PGC7s could cause tissue maceration and necrosis in banana plants.PGC7 gene is the first constitutively expressed endoPG gene isolated from Foc.4.Targeted disruption of pgc7 from Foc.The mutation of pgc7 led to a reduction of total PGs expression and regulated the expression of exo-PG genes?pgc2,pgc3 and pgc4?,revealing that PGC7 plays an important role during decomposition of pectin of host cell wall.Although the recombinant PGC7s caused banana tissue maceration and necrosis,deletion of pgc7 from Foc resulted in reduction of pathogenicity to banana cultivar‘Brazil'and‘Fenjiao'.PGC7 gene is one of the important pathogenicity genes for Foc during infection of banana. |
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