| Rice(Oryza sativa L.)is an important food crop.Meloidogyne graminicola is one of the most serious pathogens in rice production,which obviously restricts the development of rice planting and food security of China.M.graminicola maintains the presence of feeding sites by secreting effectors to the cytoplasm or apoplast of the host cells through a hollow stylet connected with the esophageal glands when infecting plants.Now,only six effectors have been studied in M.graminicola.Transcriptome sequencing of three nematode populations at different developmental stages had been finished earlier in our laboratory.In this study,seven Mg candidate effectors which were differentially expressed based on the RNA-Seq data were cloned and the expression pattern were verified by q RTPCR.The immunity suppression function of several candidate effectors was further analyzed and the secretion of candidate effector Mg＿10204 was then verified.Finally,the biological functions and pathogenic mechanisms of candidate effector Mg＿10204 were studied in depth by in vitro silencing and Y2 H.The main results of this study is presented as following:1.Seven candidate genes Mg＿36,Mg＿78,Mg＿5481,Mg＿7097,Mg＿7465,Mg＿8535and Mg＿10204,encoding C-type lectins,chorismate mutase,peptidases and unknown function,were obtained by differential expression analysis and signal peptide prediction.RNA-seq data showed that they were significantly induced at the Par-J2 stage.q RT-PCR showed that Mg＿7465 was obviously induced at the Par-J3 stage and the other candidate genes were significant up-regulated in the Par-J2 stage,indicating that Mg＿7465 may play a major role in the later development,and others may regulate Mg parasitism in the early stage.The immunity suppressive function of some candidate effector proteins was detected by PAMP and five of them were proved.2.The secretion function of Mg＿10204 signal peptide was verified by yeast and tobacco secretion system.Meanwhile,our results showed that in vitro silencing of Mg＿10204 gene significantly affect the virulence of Mg to rice.Three extracellular target proteins Os Gns3,Os EXPB6,Oryzain and one intracellular target protein Os VDAC4 of Mg＿10204 in rice were obtained by yeast two-hybrid library screening and Mg＿10204 may interact with Os Gns3,Os EXPB6,Os VDAC4 or Oryzain to promote parasitism.In summary,seven candidate effectors were obtained through RNA-Seq analysis of Mg virulent populations in this study.The expression and the immunity repression function of some candidate effectors were analyzed.Functional analysis demonstrated that Mg＿10204 could regulate nematode infection and might regulate host resistance through direct interaction with intracellular and extracellular target proteins.The candidate effectors identified in this study provide a theoretical basis for future studies on the pathogenic mechanism of M.graminicola. |
