Study On Molecular Mechanism Of SWP11 Regulating Host Defensive Reaction

Wheat blue dwarf phytoplasma belongs to the phytoplasma tentative species 16Sr I group,transmitted by Psammotettix striatus.As obligate parasites,they live strictly within plant vascular bundle cells.Wheat blue dwarf disease?WBD?,caused by wheat blue dwarf phytoplasma,is an important disease that mainly occurs in northwestern China.It can cause several symptoms in wheat,such as dwarfism and yellow leaf tips,which lead to reduced or even extinct production and seriously threatens wheat production in china.The previous study in this laboratory found that WBD phytoplasma can encode 37candidate effector proteins.The effector protein SWP11,which can cause the hypersensitive reaction in Nicotiana benthamiana,was screened by transient expression.It has great significance to prevent the proliferation and propagation of WBD phytoplasma in plants.We performed a series of assays,including bioinformatics analysis,subcellular localization and identification of host species expressing necrosis symptoms to in-depth understand the mechanism of SWP11-induced necrosis in plant.Subsequently,Nicotiana benthamiana was used as a model plant to screen and identify the targets interacting with SWP11.The main results obtained are as follows:1.Bioinformatics analysis of SWP11 were conducted.It indicated that the full length of SWP11 is 375 bp,encoding 125 amino acids.The instability coefficient of the SWP11proteinis is 50.55,and the grand average of hydropathicity is-0.650 which demonstate that SWP11 is an unstable protein and hydrophilic protein.There is a hydrophobic structure consisted in the N-terminus and the structure contains two main?-helices.There is a transmembrane region between amino acids 10-30 at the N-terminus,and a signal peptide cleavage site between amino acids 31-32.No additional transmembrane region was found after deleting the signal peptide from SWP11 protein and 93 amino acids were secreted into the extracellular.2.The subcellular localization of SWP11 and identification of host species expressing necrosis symptoms were carried out.We constructed the fusion protein expression vector p CAMBIA1302-e GFP-SWP11,and then the nuclear localization signal,nuclear export signal and nuclear export signal with loss-of-function sequence mutation were fused to the C-terminus of SWP11 to construct recombinant expression vectors p CAMBIA1302-e GFP-SWP11-NLS_SV40,p CAMBIA1302-e GFP-SWP11-NES,and p CAMBIA1302-e GFP-SWP11-NESKO.These constructions were transformed into Agrobacterium tumefaciens strain GV3101 and then inject Nicotiana benthamiana to observe its localization in cells.The results indicated that SWP11 is localized in both the nucleus and cell membrane and it can cause necrosis in Nicotiana benthamiana when SWP11 is localized on the cell membrane.Agrobacterium tumefaciens containing the p GR107-SWP11 plasmid was injected into plants,and it was found that SWP11 can induce hypersensitive reaction in tomato,pepper,tobacco,and arabidopsis.3.Using AP-SWATH and yeast two-hybrid technology to screen the targets interacting with SWP11 in Nicotiana benthamiana.The AP-SWATH?Affinity purification Sequential Window Acquisition of all Theoretical fragmentions?technology was used to screen SWP11targets and 165 proteins were obtained.The bait vectors p BT3-SUC-SWP11 and p BT3-STE-SWP11 of the split-ubiquitin membrane system were constructed and then validated that both of the vectors could be used for screening yeast library.Whereafter,87interacting proteins were obtained by using p BT3-SUC-SWP11 to screen library of Nicotiana benthamiana.The results of the screening of interaction proteins by the two methods are very similar.The targets are chlorophyll binding proteins,chloroplast photosystem I and II member subunits,ribosomal proteins,cytochrome proteins,heat shock proteins,ATPase subunits,peroxidase,carbohydrate metabolism enzymes,phosphate sugar metabolism enzymes,and amino acid-t RNA ligase or metabolic enzymes.4.Two target proteins,Nb Prx03 and Nb Prx63,which related to hydrogen peroxide metabolism were selected for the further analysis.The yeast two-hybrid assay was used to confirm the interaction of the two targets and SWP11.Bioinformatics analysis softwares and online database were applied to analyze the characteristic of sequences.The results showed that although they all belong to heme peroxidases,which is part of the non-animal peroxidase superfamily Class?,but Nb Prx03 and Nb Prx63 have a low homology rate.They have similar functions including heme binding function,metal ion(Ca²⁺)binding function and peroxidase activity,They are all involved in the catabolism of hydrogen peroxide and the response to oxidative stress,although there is a very small difference existed in functional sites.Both of them have signal peptides,of which the N-terminus of Nb Prx03 has a transmembrane region that coincides with the signal peptide while Nb Prx63has no transmembrane region.Both of the localization sites are all extracellular,which is consistent with most CIII Prxs.