Comparative Analyses Of High-throughput Sequencing Datasets And Target Genes Mining For Several Important Plant-nematodes

With a wide host range and tremendous destruction,plant parasitic nematodes cause estimated annual losses of $150 billion to world economy and their management is problematic.As the top-destructive phytonematodes,the cyst nematodes,root-knot nematodes and pinewood nematodes are threatening human food security and ecology environment in China.We performed genome and transcriptome sequencing of several nematodes from these three families,and made comparative analyses for further mining potential effectors and lethal genes.This work provides valuable resources for in-depth understanding the parasitism and pathogenicity of phytonematodes,as well as developing new targets-oriented strategies on effective and safe managements.The main results are as followings:(1)Two mixed-stages transcriptomes(pre-parasitic stages:egg,J1 and hatched-J2;post-parasitic stages:parasitic-J2,J3,J4 and adults)of Heterodera avenae were sequenced by 454 FLX+/Roche platforms.This yielded 1,066,719 raw reads(551,935 reads from pre-parasitic library and 514,784 reads from post-parasitic library),for a total 521 Mb,with an average length of 488 bp.Combined assembly resulted in 10,811 aligned contigs(with an average length of 1440 bp)and 71,401 singletons ESTs covering the whole-life stages of this nematode.Comparative analyses of the 12,703 orthologous families and phylogeny tree among H.avenae and other 7 nematodes with varied life styles revealed the significance and peculiarity of neurological system for sedentary phytonematode.The orthologous of genes involved in C.elegans immune response,diapause pathway,neurotransmitter biogenesis pathways and RNAi pathway were identified and compared among H.avenae and other nematodes.After blast against all the available EST sequences of nematoda in public databases,15.4%contigs were considered unique in H.avenae.There were 3,377 significantly differentially expressed contigs between the pre-and post-parasitic libraries.The crosstalk map of KEGG pathways enrichment demonstrated that the nervous/sensory system,environmental information processing,digestive system,circulatory system and human disease pathway were highly related and active at pre-parasitic stage;while the metabolism,genetic information processing,cell cycle and cell growth and death pathway were significantly enriched at post-parasitic stage.qRT-PCR assays validated the highest expression of mRNAs for 6 FMRFamide-like peptides at the hatched-J2 stage.For mining of effectors,we got 18 carbohydrate-active enzymes and a number of homologues to 59 reported phytonematode key genes in H.avenae.Notably,it is the first report of potential RxLR motif effectors(total 61)and genes with homologies to ANT-5/ANT-34 in phytonematodes.In addition,5 candidate effectors were chosen for preliminary experiments.Two of them were localized in the esophageal gland.Two RxLR putative effectors could suppress BAX-triggered plant programmed cell death.Interestingly,two 14-3-3 protein and a homologue of flagellum-like protein might enhance the virus pathogenicity after PVX-mediated constitutive expression in Nicotiana benthamiana.At last,1,734 genes of H.avenae screened out from the orthologous to C.elegans with RNAi lethal phenotype are considered as potential targets for developing nematode-resistant transgenic plants.(2)Three genomes of Bursaphelenchus taxa(pinewood nematode),including two pathogenic strains of B.xylophilus and a non-pathogenic but closely related species B.mucronatus,were sequenced and compared for their secretomes.The assembled genome sizes were 79.2 Mb,75.9 Mb and 69.8 Mb,respectively(with scaffold N50>350 kb and GC%? 40%).The numbers of predicted proteins in secretomes(genomes)were 2,446(17,066),2,348(17,343)and 2,209(16,137),respectively.For the secretomes,the average length of secreted signal peptides was 19aa in all strains,and 30%of proteins were assigned by Pfam domains.There were 94,88 and 95 secretory proteins with nuclear localization signal in the three nematodes,respectively.The ortholog analysis was carried out among 3 strains and resulted in 2,111 multipara-clusters of secretory proteins.Of these orthologs,408 OrthoClusters exclusive in two pathogenic strains and 414 unique proteins in the more pathogenic strain are considered as candidate effectors.(3)Through exploring publically available genomes datasets,RNAi targets with lethal phenotypes of two root-knot nematodes(Meloidogyne incognita and Meloidogyne hapla)and soybean cyst nematode(Heterodera glycines)were predicted for genetic engineering on their host plants.There were 262,306 and 253 potential targets identified in these nematodes,respectively.Of them,14 targets are shared by Meloidogyne spp.