Molecular Interaction Mechanism Between Two Genes LeMYB330 And MiPDCD6 Related To Giant Cell Apoptosis Of Root Knot Caused By Meloidogyne Incognita In Tomato

Root-knot nematode,an important pathogenic nematode,causes huge economic losses to agricultural production.The second-stage juveniles of root knot nematode invade root of host plants,then inject secretions of esophageal gland through their stylet,which stimulate the formation of giant cells and thus root knots.Some research results show that the proteins synthesized in the esophageal glands play an important role in the interaction of root-knot nematode with host plant.The induced giant cells were found vacuolation and the density of the giant cells contents were reduced gradually when the nematode begans to spawn,so it is thought that females inject proteins causing the intrinsic apoptotic pathway in the host.In this paper,we used bimolecular fluorescence complementation technology to study the protein interaction and the binding site between LeMYB330 from tomato and MiPDC D6 from RKN,and studied the silencing effect of VIGS tomato tissue culture,then analyzed their relationship with apoptosis and the apoptosis pathway they participate in the process of giant cells vacuolation in the host.In this paper,the innovative results were as follows:1?The subcellular localization of LeMYB330 and MiPDCD6 genes was clarified.We located the expression sites of protein LeMYB330 and MiPDCD6 by using the gene gun.The recombinant vector of GFP-PDCD6 and GFP-MYB330 were bombarded into onion epidermal cells,Le MYB330 protein was detected obvious green fluorescence signal in the nucleus at 487 nm green excitation light and 10×fluorescence microscope.Indicating that expression site of this protein is in nucleus.MiPDCD6 protein was detected obvious green fluorescence signal in the cytoplasm and nucleus,indicating that expression site of this protein is cytoplasm and nucleus.2?The interaction between LeMYB330 and MiPDC D6 protein was demonstrated by BiFC technique.The recombinant vector of pUC19-PDCD6 and pUC19-MYB330 were bombarded into onion epidermal cells,LeMYB330 and MiPDCD6 protein was detected obvious green fluorescence signal in the nucleus at 487 nm green excitation light and 10×fluorescence microscope.This research indicates that LeMYB330 and MiPDC D6 interacts with each other in nucleus.3?The binding site between LeMYB330 and MiPDC D6 protein was further determined.By studying the interaction between MiPDCD6??15-27aa?and MiPDC D6??82-94aa?with the full length of LeMYB330,the interaction fragment of MiPDC D6 was the first Ca²⁺binding domain.By studying the interaction between LeMYB330??9-61aa?,LeMYB330??62-116aa?and LeMYB330??9-116aa?with the full length of MiPDC D6,the interaction fragment of LeMYB330 was the R2R3 structural domain.4?The silencing effect of VIGS tomato tissue culture seedlings was ascertained.The Silencing Seedlings of VIGS-mediated MiPDCD6 and LeMYB330 were preserved by leaf tissue culture technology.Tissue culture seedlings'RNA was extracted for TRV detection,the results of electrophoresis showed that TRV virus in leaves and roots,and the roots have more virus than the leaves.The expression of LeMYB330 and MiPDC D6 was detected by qRT-PCR,The results showed that the silencing effect disappeared.