| Root-knot nematode (RKN, or Meloidogyne spp.) is a large kind of plant-parasite nematodes, which can parasite in the plant root, form galls and cause a great loss in the economic crops. Current study of RKN focuse on two part: one is about the parasitic mechanism of RKN, such as the parasitism genes and the effect proteins, which will promote our understanding of the nematode gene regulation and choose appropriate target gene for nematicide; another is about the ways to control the nematode, the primary study is the ideal biological control agents.As the development of the new generation sequencing technology, more genomes of species were sequenced, bioinformatics tools will be a powerful approach to analyze the diffrenece of the genomes, annotate the gene, identify new special gene and analyze the transcriptome. This study was divided to two party: one party is analyzing the Meloidogyne incognita effector Misp12 by bioinformatics and verificating its function;another party is revealing the specific adaption of the nematophagous Purpureocillium lilacinum 36-1 and the genes involving in the fungi parasitizing nematode-eggs.Secreted effectors in plant root-knot nematodes (RKN, or Meloidogyne spp.) play key roles in its parasite processes. Currently identified effectors mainly focus on the early stage of the nematode parasitism. There are few reports about effectors related to the latter stage. In this study, we identified a potential RKN effector gene Misp12 functioning at latter parasitism. Misp12 was 1202-bp in length, encoding a deduced protein of 149 amino acids. It was unique in Meloidogyne spp., and highly conserved in M. incognita.Misp12 encodes a secretory protein that specifically expressed at the dorsal esophageal gland by in suit hybridization, and highly up-regulated at female stages by developmental expression analyses. Transient expression of the Misp12-GFP protein in onion epidermal cell showed that Misp12 was localized in cytoplast. Using the VIGS approach, we found that the RNAi of Misp12 could significantly affected the galls formation, numbers of eggs and nematodes. It revealed that in planta RNA interference targeting Misp12 suppressed the expression of Misp12 in nematodes and attenuated latter parasitic ability of M.incognita. Furthermore, up-regulation of JA and SA pathway defensive related genes in Misp12-silenced plant indicated the gene might be associated with the suppression of plant defensive response. Expressing Misp12 in Nicotiana benthamiana could suppress programmed cell death triggered by INF1 (PT-PCD). Additionally, the expression level of SA marker genes PAL5 and PR1 were were strongly induced in control plants and were repressed in plants overproducing Misp12Purpureocillium lilacinum is believed to be a promising nematophagous ascomycetes living in diverse environments. It was also found to be an opportunistic fungi on humanity.A commercial product has been registered to control plant parasitic nematodes. But the molecular mechanism on the toxicological process is still ambiguous due to relative few reports. Using Illumina paired-end sequencing, the draft genome sequence of P. lilacinum strain 36-1 and transcriptom of this fungi infection nematode-eggs were determined, we found P. lilacinum strain 36-1 was more closely related to O. sinensis, an obligate parasitism fungus for insect. Based on the langley-fitch analysis, we found that P.lilacinum strain 36-1 diverged before a split with O. sinensis 74?94 million years ago(MYA). Whole genome alignment indicates that P. lilacinum 36-1 possess a more dynamic genome in comparison with P. lilacinum India strain. However, more genome segments rearrangements and SNPs indicated that the genome of P. lilacinum 36-1 was more dynamic than the India strain. The P. lilacinum strain 36-1 had a weak RIP, and it went through little selective pressure during the process of evolution by Ka/Ks analysis.Analyzing the protome of the P. lilacinum, we found over-representative of serine peptidases, chitin degradation related genes, transporters and transduction signal genes in P. lilacinum than other fungi, these proteins may play a critical role during the early stage of the fungus infection nematode-eggs. The expression of appressorium forming and antioxidant related genes exhibited the similar infection patterns in P. lilacinum strain 36-1 as those of the model entomophagous fungi Metarhizium spp.. Analyzing the transcriptome of the P. lilacinum, the gene ontology annotation (GO) enrichment result showed that the up-expressed genes related to oxidoreductase activity, structural constitute of ribosome, transferase activity and transmembrane tranporter activity were enriched.Consistently, KEGG enrichment showed that up-regulated genes were mainly enriched in energy, amino acid and lipid metabolism. These results suggested that P. lilacinum might undergo obvious enegy metabolic processes turnover during early stages of nematode-eggs infections. |
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