Cloning And Functional Analysis Of Mj-msp33 From The Root-Knot Nematode Meloidogyne Javanica

The root-knot nematode(Meloidogyne. spp) belonging to sedentary obligate Plant-parasitic nematodes, can secrete multiple effector proteins which engage in the interaction between plant and nematode during infection. These effector proteins act on a variety of different plant defensive pathway to facilitate infection, development and reproduction, so that to maintain a dynamic equilibrium symbiosis between nematode and host plant. The esophageal gland of root-knot nematode can secrete proteins to esophageal, which have N-terminal signal peptides, then beening injected into host tissue via a syringe-like stylet. Studing these effector proteins' functions will increase our knowledge about the molecular mechanism of the nematode-plant interactions, in turn to supply reliable theory bases to nematode control.In this study, we have cloned a parasitic related gene Mj-msp33 from Meloidogyne javanica by homo-clone, based on Mi-msp33 partial cds of Meloidogyne incognita, and get its overall c DNA sequences using RACE technology. The Mi-msp33 has only 333 bp c DNA sequences at N-terminal. What we have obtained is that the gene Mj-msp33 of M. javanica have a 1326 bp open reading frame(ORF) which encode a 442 aa protein.The Mj-msp33 protein with a N-terminal signal peptide is a novel candidate effector protein of M. javanica. It possesses a pat4:KKHK nuclear localization signal(NLS) and has no homologous proteins. To identify the functions of Mj-msp33, we have proceeded In-situ hybridization, Developmental expression analysis and Yeast two-hybrid.For In-situ hybridization, we used a Mj-msp33-specific probe labeled by Digoxin. The result shows that Mj-msp33 is localized at subventral esophageal gland of M. javanica. We analyzed developmental expression patterns of Mj-msp33 by q RT-PCR. It reveals that Mj-msp33 get the highest expression at pre-J2 and 2dpi(day post inoculation). When nematode sets up its unique feeding site, the initiation of a gaint cell which contains a multiple nucleus, the expression of Mj-msp33 is gradually down-regulated along with nematode's development.For Yeast two-hybrid, we have screened a protein of Arabidopsis thaliana, glycerophosphodiester phosphodiesterase(GDPD), which may interact with Mj-msp33, using a fusion protein as bait. The A. thaliana GDPD regulates the structure of the cell wall and involves in perception of lipid-derived molecules from microbial pathogens or damage responses.According to these results, we can conclude that Mj-msp33 is a secreted protein of M. javanica, which functions may be related to nematode's parasitism, and plays an essential role on nematode-plant interaction.