The Development Of Nematode Feeding Sites And Function Of MPK5 During Meloidogyne Graminicola And Rice Interaction

Meloidogyne graminicola is an important soilborne pathogen of rice,leading to severe yield loss,Meloidogyne spp.establish feeding sites(NFSs)-giant cells(GCs)inside host roots by secreting effectors and hijacking host genes to ensure its nutrient uptake.In addition,mitogen-activated protein kinase(MAPK or MPK)cascades are widely involved in biological processes of plant growth,development and stress responses.MPK could be activated through complex signaling pathways,which then phosphorylate their target proteins to coordinate different cellular responses,converting biotic and abiotic signals into appropriate cellular responses and interacting with many signaling pathways.Until now roles of MAPK cascades in interaction mechanisms between plants and nematodes are yet unknown.In order to address these two conundrums,we characterized differences of the spatiotemporal three-dimensional(3D)structures and cytoplasmic densities of M.graminicola-caused NFSs on rice by tissue transparency treatments and cell 3D-structure reconstruction technology to elucidate the potential developmental process of M.graminicola-caused NFSs.Meanwhile,both over-expression and gene silencing rice lines of rice MPK5 were used as plant material,the effect of imbalance MPK5 homeostasis on rice-M.graminicola interaction was evaluated,and the potential mechanism of MPK5 mediated rice resistance to M.graminicola was investigated.The main conclusions are shown as follows:1.Complete serial optical sections of M.graminicola-caused NFSs at each developmental stage were obtained by confocal microscope,and 3D structures of these NFSs were reconstructed through the software Fiji.Differences in 3D structures,volume sizes as well as lateral and longitudinal areas among M.graminicola-caused NFSs at later parasitic second stage(later p-J2),J3,J4 and adult female stages were characterized.Results indicated that three types of M.graminicola-caused NFSs,type I,type II and type III,could be distinguished based on the difference in 3D structures of NFSs.Type I is the majority at all stages tested,accounting for 45.5%,84.4%,88.9%and 90.2%at later p-J2,J3,J4 and adult female stage,respectively,whereas type III accounts for the least.Type II develops into type I at J3 stage by its longitudinal growth.In addition,the lateral and longitudinal expansion ceases at later p-J2 stage and the non-feeding J4 stage,respectively with the average volume size of NFSs at J4 stage being approximately 2.69×10~6μm~3.2.Differences in cytoplasm density among M.graminicola-caused NFSs at different stages were assessed through using the software Fiji to compare strengths of green fluorescence signals of optical sections of NFSs tested.Data showed that cytoplasm density of M.graminicola-caused NFSs reaches a peak at the midpoint of early p-J2 and adult female stages with the highest one at adult female stage.Our data suggest that the NFS polarized expansion stops before full NFS maturation,implying NFS expansion does not synchronize with the maturation of NFSs.3.Using both MPK5-overexpression and-silencing rice lines as plants,M.graminicola-caused galls and nematode inside roots on these two transgenic rice lines reduced compared with rice wild-type according to the data from nematode inoculation test,where at 3 days post inoculation(dpi)the average numbers of galls and nematodes inside roots per root system reduced by that in MPK5-overexpression line being from110to 1 and 0 as well as from 22 to 0,respectively,and from 13 to 1 and 2 as well as from 25 to 1 and 4,respectively in MPK5-silencing line at 10 dpi.These data suggest that imbalance of MPK5 homeostasis in rice could result in impeding susceptible interaction between rice and M.graminicola.4.Results of qRT-PCR analysis showed that different from rice wild type MPK5was induced by M.graminicola in both MPK5-overexpressing and-silencing rice lines,where the relative expression level was 41.8 and 9.0,respectively.Meanwhile,M.graminicola inhibited the expression of chitinase gene in MPK5-overexpressing lines,but induce the expression of disease-related genes PR5 and PR10 in MPK5-silencing lines.These data indicate that the imbalance of MPK5 homeostasis could make MPK5response to M.graminicola infection,and the mechanism of MPK5-mediated resistance to M.graminicola in rice is different between MPK5-overexpressing and-silencing rice lines.