| Radopholus similis is an important migratory endoparasitic nematode.It is mainly distributed in the tropical and subtropical regions and greenhouses of temperate regions.It harms banana,citrus,pepper and various ornamental and economic crops.Little is known about the molecular mechanisms of the infection and pathogenesis of R.similis.In order to understand the developmental,reproductive and parasitic characteristics of Radopholus similis at the molecular level,the transcriptome of R.similis eggs,juveniles,females,and males was sequenced separately for the first time using de novo sequencing with the Illumina Hi Seq TM 2000 platform.Based on the transcriptome data of R.similis,five pectate lyase(pel)genes and a serine carboxypeptidase coding gene Rs-scp-1 were further analysied with RNAi.The main results of this study were listed as follow:1.The transcriptome data of R.similis eggs,juveniles,females,and males were obtained using de novo transcriptome sequencing.51,981,55,176,45,129 and 39,599 unigenes were obtained from these four life stage of R.similis respectively.Gathered these four libraries together,a total of 64,617 transcripts were obtained.48,432 unigenes were annotated in five databases.A total of 54,066 predicted coding sequences(CDS)were identified,of which 47,895 CDSs were annotated.The analysis of transcript expression differences between eggs and juveniles showed that 6,159 transcripts were differentially expressed,of which 2,613 were up-regulated and 3,546 were down-regulated.There were 2,218 differentially expressed transcripts between juveniles and females,of which 1,350 transcripts were up-regulated.5,649 transcripts were differentially expressed during juvenlies development into males,including 3,214 up-regulated transcripts and 2,435 down-regulated transcripts.The functional genes in R.similis were predicted,including 86 putative cell wall degrading enzymes,102 putative antioxidase genes and 109 putative genes that may be involved in parasitism progress.At the same time,the transcripts involved in nematode chemotaxis(2,442 transcripts)and RNAi(273 transcripts)were also predicted.This study also found that 744 transcripts of R.similis were highly similar to the transcripts of the host citrus gene sequence,of which 741 transcripts were only expressed in juvenlies and three transcripts were expressed in juvenlies and females.GO analysis showed that these transcripts were mainly involved in the processes of organic metabolism,cell metabolism,synthesis of substances,and synthesis of nitrogenous compounds.2.The pectate lyase genes Rs-pel-1,Rs-pel-2,Rs-pel-3,Rs-pel-4,and Rs-pel-5 were cloned based on the transcriptome data of R.similis.The ORF lengths of the Rs-pel-1,Rs-pel-2,Rs-pel-3,Rs-pel-4,and Rs-pel-5 were 843,819,873,771,and 810 bp,respectively.The encoded amino acids are 280,272,290,256 and 269 aa in length,respectively.All five R.similis PEL proteins were extramembrane proteins,of which Rs-PEL-2,-4,and-5 having signal peptide sequences,and Rs-PEL-1,Rs-PEL-3 do not have a signal peptide.In situ hybridization showed that five pel genes in R.similis were all located in the esophageal gland.q PCR showed that the expression levels of Rs-pel-2,Rs-pel-3,Rs-pel-4,and Rs-pel-5 were higher in juvenlis than in other life stages.The expression levels of Rs-pel-1 were highest in females,followed by males,juvenlis and eggs.3.RNAi of Rs-pel-1,-2,-3,-4,and-5 were performed using ds RNA soaking.The result showed that the expression lever of these five genes were significantly decreased(p<0.05),and the expression levels of the five genes were minimized after soaking for 24 h.Nematodes were then inoculated into carrot disks after RNAi treatment.Compared with water treatment,R.similis treated with Rs-pel-1,-2,-3,-4,and-5 ds RNAs showed reduced number.The expression levels of Rs-scp-1 were significantly suppressed after R.similis was soaked in Rs-scp-1 ds RNA for 12 h.Nematodes were then inoculated into Anthurium andraeanum after RNAi treatment.Compared with water treatment,R.similis treated with RNAi showed reduced number and pathogenicity.In this study,the life-stage specific transcriptome was performed in migratory endoparasitic nematode,Radopholus similis for the first time.The differences in species,number,and developmental expression patterns of parasitism gene between migratory and sedentary endoparasitic nematode were revealed.For the first time,a large number of transcripts that were horizontally transferred from host plants were found in plant-parasitic nematodes.Based on transcriptome data,five pectin lyase genes located in the esophageal glands were cloned,revealing the diversity of gene types and expression patterns of pectate lyase in migratory endoparasitic nematode for the first time.The RNAi of the five pel genes and Rs-scp-1 confirmed the relationship between these genes and the pathogenicity of R.similis.The analysis of the transcriptome of R.similis and identify of these parasitism gene will help to understand the molecular pathogenic mechanism of R.similis and find a new target for the prevention and cure of R.similis. |
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