Transcriptome Analysis Of Mixed Stages Of Meloidogyne Enterolobii And Identification Of Four Candidate Parasitism Genes

Meloidogyne enterolobii, as a kind of root-knot nematode (RKN), is an important obligate biotrophic plant parasite that is resistant to high temperature and has strong pathogenicity within a wide host range. Meloidogyne enterolobii can overcome nematode resistant genes such as tomato Mi and pepper Me. It causes great economic loss to crop production, and thus has been regarded as an important quarantine object around the world. We extracted the total RNA from the mixed stage of M. enterolobii, sequenced its transcriptome using Roche GS FLX sequencing system and obtained 408 663 reads that were assembled into 8 193 Contigs and 31860 Singlets with the averaged lenghth as 961 bp and 416 bp, respectively.8 143 Contigs and 29 043 Singlets had predicative open reading frame (ORF), of which 4 521 ORFs were GO annotated.The ortholog analysis was carried out by comparing M. enterolobii with several orthologous nematodes that had available genetic information, including M. incognita, M. hapla, B. xylophilus, G. pallida and C. elegans. Phylogenomic analyses using a concatenated set of 1 022 single-copy ortholog families conserved in all species revealed that M. enterolobii was most closely related to M. incognita. We subdivided these orthologous families into five groups and analyzed the gene families that were not shared by all these nematodes. The results showed that nine of the functional GO subgroups existed in the three Meloidogyne species, two GO subgroups existed in M. enterolobii and non-Meloidogyne species, one GO functional subgroup existed in both M. incognita and M. enterolobii and eleven were in both M. hapla and M. enterolobii. KEGG pathway analyses showed that eight pathways were unique to M. enterolobii, including tetracycline biosynthesis, beta-lactam resistance, bisphenol degradation, peptidoglycan biosynthesis, atrazine degradation, steroid degradation, biosynthesis of the siderophore group nonribosomal peptides, and malaria. The number of pathways that were unique to M. incognita and M. hapla were five and one, respectively. Compared with the other nematodes, three pathways had the highest KOs in M. enterolobii, while forty-one pathways were missing in M. incognita. From our analysis using SignallP and TMHMM, M. enterolobii was found to have 1 464 putative secretory proteins. Venn diagram of three closedly related nematodes showed that 701 predicted secretory proteins belonged to M. enterolobii, while 679 were conserved in all three species. We comparatively analyzed the transcriptomes in different nematodes including the transcripts that putatively coded for CAZymes, kinases, neuropeptide genes and secretory proteins and those that were involved in the RNAi pathway and immune signaling, all of which were potential targets for biological control.We also found 241 transcripts from M. enterolobii that could be translated into effectors as reported. These putative effectors were involved in cell wall degradation, proteinase, defense or suppression of the host plant immune system, participation in the signaling of the host plants and other similar functions. Based on these information, we cloned four candidate proteins that were secreted from the esophageal gland cells with signal peptides. We preliminarily examined these four proteins using a PVX (pGR107)-based high-throughput transient plant expression system in Nicotiana benthamiana (N. benthamiana). The results showed that No.5 and No.10 proteins effectively suppressed BAX-induced PCD and No.8 protein caused necrosis, whereas No.7 protein failed to suppress BAX-induced PCD or cause necrosis. To determine the localization of four candidate effecotrs in plant cells, a protein transient expression assay was performed. The results showed that three candidate effectors would be mainly imported into the plant cell nucleus following delivery into the cytoplasm of host cells by the nematodes, while the other one was mainly expressed in the plasma membranes. These results have laid a foundation for further in-depth study of new effectors in the potent pathagenenic nematode.