| Litchi downy blight is the most destructive disease in litchi production,storage and transportation.The cause agent,Peronophythora litchii,is belongs to Chromista and Oomycota.At present,little is known about the molecular pathogenesis of this pathogen.Thus,in order to understand the changes of gene expression in the infection process macroscopically,this study attempts to use high-throughput sequencing to analyze the zoospores and the infection stages of this pathogen.Subsequently,based on the Nicotiana benthamiana system,a large-scale immune response screening of RXLR effectors,an important family of secretory proteins,was carried out.The biological functions and recognition mechanism of the selected effector proteins were further studied.The main results were as follows:1.By the RNA-Seq of Zoospore(Zo),1.5 hpi(hour post inoculation),3 hpi,6 hpi,12 hpi and 24 hpi,there were 43321179-53216790(96.04%-97.09%),195591-480054(0.24%-0.54%),275192-817712(0.26%-0.80%),625730-1460797(0.68%-2.89%),2864082-9805140(3.03%-9.83%)and 4593420-27613116(5.48%-27.06%)reads could mapped to the P.litchii genome.2.Differential expression gene analysis showed that 1784 genes were up-regulated and1987 genes down-regulated at 1.5 hpi,2330 genes were up-regulated and 2702 genes down-regulated at 3 hpi,2843 genes were up-regulated at 6 hpi,and 3411 genes were up-regulated at 6 hpi.There were 3426 genes up-regulated and 3619 genes down-regulated12 hpi,3739 genes up-regulated and 3269 genes down-regulated 24 hpi.GO enrichment analysis of these differential expression genes showed that the up-regulated genes mainly belonged to biological process and were most concentrated in oxidation-reduction process.KEGG analysis showed that NADPH: quinone reductase(K00344)pathway,glutathione S-transferase(K00799)pathway,2-methylene-furan-3-one reductase(K18980)pathway,pectinesterase(K01051)pathway,alkaline phosphatase(K01113)pathway,beta-glucosidase(K01188)pathway,serine/threonine-protein kinase TNNI3K(K17535)pathway pathway,phospholipid-transporting ATPase(K14802)pathway and the nuclear protein localization protein 4 homolog(K14015)pathway are the main biochemical pathways in DEGs.3.There were 179 RXLR genes were expressed in different stages via the transcriptome analysis.212 RXLR genes were cloned and constructed into PVX vector.Three of them,Pl Avh23,Pl Avh133 and Pl Avh142,could induce cell death in N.benthamiana by Agrobacterium tumefaciens mediated transient transformation.In addition,it was found that Pl Avh41,Pl Avh43,Pl Avh69,Pl Avh104 and Pl Avh115 could stably inhibit the cell death induced by Pl Avh142.Transcriptome data analysis showed that these five effector protein genes were significantly up-regulated in the infection stage4.Pl Avh142 could induce broad-spectrum cell death in N.benthamiana,S.melongena and S.lycopersicum.Transient expression of Pl Avh142 could increase the accumulation of reactive oxygen species and callose in plants,and activate the pathways of salicylic acid,jasmonic acid and ethylene.Polymorphism analysis showed that this effector gene was conserved in 30 strains of P.litchii collected from different years and regions.Only 5 SNP of Pl Avh142 were found in this 30 strains and three of which resulted in non-synonymous substitution,but all the three versions of PAvh142 could cause cell death in N.Benthamiana.5.Subcellular localization studies showed that Pl Avh142 localized in both the nucleus and cytoplasm of plant cells,and the cytoplasmic localization was sufficient to induce cell death.Conserved domain prediction shows that there are two internal repeats at the C-terminal,and the absence of any internal repeat will abolish its cell death-inducing activity.In addition,when RAR1,SGT1 and HSP90 genes in N.Benthamiana were silenced by VIGS,the cell death-inducing activity of Pl Avh142 significantly decreased or disappeared.6.RT-q PCR analysis showed that Pl Avh142 was up-regulated in zoospore and infection stages.The expression peak appeared at 3 hpi,and then rapidly declined onward.Pl Avh142 gene was successfully knocked out by CRISPR/Cas9 gene editing system.Subseqently,the overexpression mutants were also obtained.The results of pathogenicity assay showed that Pl Avh142 knockout mutants were less virulent on litchi and Pl Avh142 overexpressing lines were more virulent.7.It was found that there were several similar copies of Pl Avh23 gene in the genome of P.litchii.But,only Pl Avh23-2 has a typical RXLR motif.Both Pl Avh23 and Pl Avh23-2 could induce cell death in N.benthamiana.Transient expression of Pl Avh23-2 also increased the accumulation of reactive oxygen species and callose,and activated the pathways of salicylic acid,jasmonic acid and ethylene.Through the VIGS assay,cell daeth triggered by Pl Avh23-2 depends on SGT1 and HSP90 of plants.Above results provide an important basis for the discovery of pathogenic genes,and lay a solid foundation for finding disease resistance genes and understanding the role of RXLR effector proteins in the pathogenesis of this pathogen. |
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