Function And Mechanism Characterization Of Gma-miR1510 And GmWRKY40 In The Interactions Of Phytophthora Sojae And Soybean

Phytophthora root and stem root(PRR)caused by Phytophthora sojae is one of the devastating diseases that seriously threat soybean production.In recent years,PRR was gradually expanding,and caused significant economic losses in China's soybean main production regions.The most effective approach to reduce losses caused by P.sojae is the use of genetic resistance and tolerance in soybean.However,the variability of P.sojae is very high,and resistant cultivars will lose their resistance with the new race population increasing and toxicity changing.Therefore,the study of molecular mechanism of interaction between soybean and P.sojae is one of the fundamental ways to solve the problem.Plants have gradually formed and improved a series of complex and effective protection mechanisms and defense networks in the development process in order to resist the infection of pathogens.Plant miRNAs play important roles in plant defense responses by post-transcriptional regulation of target genes.gma-miR1510 is one of the miRNAs that response to P.sojae infection detected by microarray analysis.The predicted target genes of gma-miR1510 encode NB-LRR type disease resistance proteins.In this study,the target gene of gma-miR1510 was identified by 5'-RACE and the function of gma-miR1510 was analyzed by overexpression in soybean hairy roots.WRKY transcription factors is one of the largest families of transcriptional regulators in plants and form integral parts of signaling webs that modulate many plant processes,which play a key role in plant disease resistance.In this study,GmWRKY40 was silenced by RNA interference.The function in the soybean response to P.sojae was elabrated.The main results are as follows:1.Function characterization of gma-miR1510 in the resistance of soybean to P.sojae(1)The expression of gma-miR1510 is reduced upon P.sojae infectionThe gma-miR1510 abundance after infected with P.sojae was detected by stem-loop qRT-PCR.The expression level of gma-miR1510 was down-regulated gradually by P.sojae treatment.It suggest a potential role of gma-miRl 510 in the soybean defense against P.sojae.(2)Glyma.16G135500 is identified as the target gene of gma-miR1 510Most of the predicted target genes of gma-miR1 510 encode NB-I.LRR-type disease-resistant proteins.Glyma.16G135500 was identified as the target of gma-miR1510 by 5'-RACE,which encode typical plants disease resistance TIR and NB-LRR domain.gma-miR1510 was negatively correlated with the expression of Glyma.16G135500 during the interaction between soybean and P.sojae.The expression of Glyma.16G135500 was significantly inhibited in the hairy root overexpressed gma-miR1510a/b.These suggest that Glyma.16G135500 is targeted by gma-miR1510.(3)The stress response cis-regulatory elements were distributed in the promoters of gma-MIR1510 and Glyma.16G135500The PlantCARE database was used to analysis the cis-acting elements in the promoters of gma-miR1510a and Glyma.16G135500· We found there are some fungal elicitor response,defense and stress response and multiple hormones response cis-acting elements,suggesting that it was likely to be involved response of soybean to pathogens.(4)Overexpression of gma-miR1510a/b reduces soybean resistance to P.sojaeTransgenic roots were inoculated with zoospore suspensions of P6497R,and the number of roots with hyphae successfully infected and oospore development at the inoculated area was observed and counted.Compared with control,the gma-miR1510a/b overexpressed hairy roots exhibit sensitive to P.sojae.Transgenic roots overexpressing gma-miR1510a/b exhibited a greater P.sojae biomass accumulation compared with the control.It demonstrate that gma-miR1510a/b negatively regulate the soybean defense against P.sojae.2.Function characterization of GmWRKY40 in the resistance of soybean to P.sojae(1)The expression of GmWRKY40 is induced by P.sojae and various exogenous hormonesThe results of qRT-PCR showed that the expression of GmWRKY40 was up-regulated after treated with P.sojae and exogenous hormones(SA,MeJA,ETH and ABA),indicating that GmWRKY40 may be involved in plant response to pathogens by regulating plant hormone signaling pathway.(2)GmWRKY40 located in the nucleusGFP fusion expression vector pBIN-WRKY40-GFP was constructed and transient expression it in tobacco leaf by way of Agrobacterium tumefaciens mediation,showing that GmWRKY40 protein is mainly distributed in the nucleus.(3)GmWRKY40 participates in the soybean basal resistance to P.sojaeSilencing GmWRKY40 in Williams(rps)showed that the lesion length of P.sojae infection was significantly longer than that of control.Transgenic roots silencing GmWRKY40 exhibited a greater P.sojae biomass accumulation compared with the control,indicating that GmWRKY40 involved in the basal resistance of soybean.(4)Silencing GmWRKY40 attenuates soybean vertical resistance to P.sojaeTransgenic roots of Williams 82(Rps 1k)were inoculated with zoospore suspensions of P6497R,and the number of roots with hyphae successfully infected and oospore development at the inoculated area was observed and counted.Compared with control,the GmWRKY40 silenced hairy roots exhibited more sensitive to P.sojae.Transgenic roots silencing GmWRKY40 exhibited a greaterP.sojae biomass accumulation compared with the control,demonstrating that GmWRKY40 involved in the soybean response to P.sojae,and silencing GmWRKY40 attenuated its vertical resistance.DAB staining and H2O2 content were analyzed in transgenic hairy roots infected with P.sojae.It showed that the accumulation of H2O2 in the GmWRKY40 silenced hairy roots was significantly lower compared with control.After inoculation,the expression of NADPHox associated with H2O2 synthesis was significantly lower than that of the control,otherwise,the expression of APX1 related to H2O2 scavenging was significantly up-regulated,indicating that GmWRKY40 participates in the accumulation of H2O2 during the infection of P.sojae.The expression of SA and JA signal pathway-related genes were analyzed in transgenic hairy roots before or after infected with P.sojae.The results showed that NPR1,a marker gene of SA signal pathway,was significantly down-regulated in the GmWRKY40 silenced transgenic hairy roots after inoculation compared to the control.The expression of other two genes PR1a and PR5 were lower than those of control before or after inoculated with P.sojae.The transcription level of PDF 1.2,a marker gene of JA signal pathway,was significantly down-regulated than that of control after infected with P.sojae.Indicating that GmWRKY40 activate SA and JA signal pathways.(5)GmWRKY40 interacts with JAZ family proteinsTwo JAZ proteins and a Zinc finger protein were identified interact with GmWRKY40 by screening yeast cDNA library.We further analyzed the interaction of JAZ family other proteins with GmWRKY40.It showed that GmWRKY40 interact with 8 JAZ family proteins.The interactions between GmWRKY40 and JAZ were inhibited differently when mutated the WRKY domain or the Zinc finger domain of the GmWRKY40 protein.