| The pathogen of Phytophthora root and stem root(RRR)is Phytophthora sojae,which is one of the devastating diseases in soybean production,caused increasing economic losses.Cultivating genetic resistance and tolerance in soybean has been an economical and effective approach to control this disease.However,due to the fast variation rate of P.sojae,it is difficult to meet the production need simply by screening the resistant soybean.Therefore,it is necessary to analyze the molecular mechanism of interaction between soybean and P.sojae,so as to fundamentally prevent and control this disease.Phytohormone plays a role in regulating plant resistance to pathogens as a natural signaling molecule.Jasmonates are recognized as a plant defense hormone,which is involved in plant-pathogen interactions by regulating the expression of defense response genes.JAZ protein is an important negative regulator of jasmonic acid signaling.The JAZ genes were up-regulated in Gm WRKY40-RNAi transgenic hairy roots after P.sojae infections by RNA-seq,as previously described.This suggested that the JAZ genes may be involved in interaction between soybean and R sojae.In this study,the function of JAZ gene in soybean resistance to P.sojae was elaborated,through bioinformatics analysis and functional analysis.The main research results are as follows:1.Identification and response to P.sojae infections of JAZ gene family in SoybeanBy bioinformatics analysis,24 JAZ genes had been discovered in the soybean genome,which were unevenly distributed on 14 chromosomes and maximum number of genes(4 JAZ genes)on chromosome 9.the JAZ gene family of soybean,Arabidopsis and rice can be divided into 5 subgroups(C1～C5)in the phylogenetic tree,which the soybean JAZ gene had the closest relationship with that in Arabidopsis.JAZ gene in the same subgroup had the number and length of exons and introns in common.Most members of this family contained jasmonic acid(JA)and abscisic acid(ABA)responsive cis-acting elements,and a few contained defense and stress related cis-acting elements.After P.sojae infections,the expression of C4 subgroup members were significantly up-regulated,the expression of C1 subgroup members were slightly up-regulated,while the expression of C2,C3 and C5 subgroup members were down-regulated.This suggests that soybean JAZ gene family members might have different expression profiles to the treatment of P.sojae.2.Functional analysis of GmJAZ17 in soybean resistance to P.sojaeG2JAZ17 was detected in four tissues by qRT-PCR,including soybean root,stem,leaf and cotyledon,with the highest expression in root,the second in leaf,and the lowest in stem and cotyledon.GmJAZ17 was induced by MeJA and its expression level peaked at 3 h after treatment,and then gradually decreased.Overexpression vector pBIN-GmJAZ17-GFP4 was constructed to obtain the fusion protein of the gene and GFP protein.By the Agrobacteriummediated transient expression system,the fusion vector was injected into tobacco leaf cells to observe the localization of GmJAZ 17 protein.The result showed that GmJAZ 17 protein was located in the nucleus.GmJAZ17 gene was overexpressed in Williams(Rps)to obtain transgenic hairy roots.After the P.sojae infections,the overexpressed transgenic hairy roots showed that the diseased spots were longer and more severe than those in the control group.After inoculation of P.sojae zoospores,the P.sojae biomass accumulation in overexpressed transgenic hairy roots was higher than that in the control group.In addition,the expression of defense-related genes GmPR1a and GmPR5 were both up-regulated in two groups,but the upregulation degree in overexpressed hairy roots was significantly lower than that in the control group.Furthermore,the expression of GmPDF1.2,a defense marker gene in the JA signal,was also inhibited in overexpressed hairy roots,compared with the control group.It suggests that GmJAZ17 may negatively regulate the resistance to P.sojae by JA signaling in soybean.GmJAZ17 was identified to interact with other GmJAZ proteins to form a homo-and heterodimerization by yeast two-hybrid.In addition.GmJAZ 17 strongly interacted with GmMYC2(highly homologous to AtMYC2),a key transcription factor of JA signaling.By screening the yeast cDNA library from soybean infected by P.sojae,GmJAZ 17 were identified to interact with other GmJAZ proteins,such as GmJAZ9,GmJAZ15,GmJAZ19,etc.,in addition to some defense-related proteins GmOLPa,GmOPLb.To further verify the interaction proteins by BiFC experiments,GmMYC2 and GmJAZ19 were constructed to the cYEP vector,and GmJAZ17 to the nYEP vector.Using the Agrobacterium-mediated transient expression system,GmMYC2-cYEP and GmJAZ17-nYEP were co-injected into tobacco leaf cells,and GmJAZ19-cYEP and GmJAZ17-nYEP were also co-injected into tobacco leaf cells.The two groups could interact with each other in tobacco cells,and yellow fluorescence signals were detected in both groups by confocal laser scanning microscope. |
