Mechanism Of Soybean MiR159 Involved In Gibberellin-modulated Resistance To Soybean Cyst Nematode

Soybean[Glycine max（L.）Merr.]plays an important role in China’s national economy.Soybean cyst nematode（SCN）is one of the most serious pathogens of soybean yield.Studies have shown that gibberellin and mi R159 respond to the process of cyst nematode parasitizing soybean.However,the specific function and mechanism of gibberellin and mi R159 in the interaction between soybean and cyst nematode remain to be elucidated.In this study,the functions of gibberellin and mi R159 in the interaction between soybean and cyst nematode were verified,and the molecular mechanism of gibberellin and mi R159 regulating soybean resistance to cyst nematode was clarified.The main results are as follows:1.Soybean mi RNA respond to the SCN infection at early stages.A total of 24s RNA libraries were constructed from resistant HPZ and susceptible W82 roots infected with soybean cyst nematode race 3 at 1 dpi and 5 dpi.A total of 634 known mi RNA and252 novel mi RNA were identified,14 known mi RNAs and 26 novel mi RNAs were differentially expressed（DE）under.SCN infection.The functional annotation of the target genes of DE mi RNAs were highly related to reactive oxygen species burst,plant hormone signal transduction and disease resistance.2.mi R159 positively regulate soybean resistance to SCN.Sequence analysis showed that there were five MIR159 genes in soybean genome,namely gma-mi R159a/b/c/e/f,respectively.The corresponding precursor sequence was pre-mi R159a/b/c/e/f,and the mature sequence was mi R159-3p.Phylogenetic analysis showed that soybean mi R159 was mainly divided into two subfamilies,gma-mi R159a/e and gma-mi R159b/c/f.Basing on plant binary expression vector p NINC2RNAi,mi R159 overexpression and STTM-mi R159 silencing vectors were constructed and used for functional verification by inducing transgenic soybean hair roots,compared with EV,the expression of mi R159 in pre-mi R159 transgenic hairy roots increased by 6-15 times,while the expression of mi R159 in STTM-mi R159transgenic hairy roots decreased to less than 0.5 times.Overexpression of mi R159decrease the number of cyst nematodes,improve the resistance of soybean and arrest the development of nematodes in transgenic hairy roots.Conversely,silencing mi R159decrease the resistance of soybean to cyst nematode.3.Gm MYB33b gene negatively regulate soybean resistance to SCN.Through online analysis on ps RNATarget and verification of 5’-RACE experiments,it was proved that mi R159 cleavage six GAMYB genes,namely:Glyma.04G125700,Glyma.06G312900,Glyma13G073400,Glyma.13G187500,Glyma15G225300 and Glyma.20G47600.Through phylogenetic and conserved domain analyses,it was found that the above six genes had closer genetic relationship with At MYB33 of Arabidopsis thaliana.Hence,these six GAMYB genes were named as Gm MYB33c,Gm MYB33d,Gm MYB33b,Gm MYB33e,Gm MYB33f and Gm MYB33a,respectively,and Gm MYB33protein was a typical R2R3 type MYB transcription factor.Under SCN infection,the expression of Gm MYB33a,Gm MYB33b,Gm MYB33c,Gm MYB33e,Gm MYB33d and Gm MYB33f in soybean W82 roots were up-regulated,and there were similar expression patterns at each time point.Functional of Gm MYB33b was verified by constructing Gm MYB33b overexpression and Gm MYB33b RNAi silencing transgenic soybean hair roots,The expression of Gm MYB33b in soybean cyst nematode inoculated roots was up-regulated by 4 times-fold compared with uninoculated W82 roots.Compared with EV,the expression level of Gm MYB33b gene was increased by 14.31 times-fold under the drive of Gmubi promoter,while the expression level of Gm MYB33b gene in RNAi silencing vector driven by Ca MV35S promoter was decreased by 0.45 folds.At 1 dpi,there was no significant difference in nematode infection among EV,Overexpression of Gm MYB33b decreased soybean resistance to cyst nematode and promoted the development of cyst nematode.On the contrary,silencing Gm MYB33b increased the resistance of soybean to cyst nematodes and delayed the development of nematodes.4.Gibberellin positively regulated soybean resistance to SCN and the expression of miR159.In the early stage of nematode infection（1 dpi and 5 dpi）,the genes regulating gibberellin metabolism up-regulated and the genes regulating gibberellin synthesis down-regulated.At the late stage of nematode parasitic process,there was no obvious opposite trend in the gene expression patterns regulating gibberellin synthesis and metabolism at 10 dpi and 15 dpi.The infection of SCN led to the decrease of gibberellin content in soybean roots,including biological active GA₁ and GA₃ with were significantly down-regulated at the early stage（1 dpi and 5 dpi）of nematode infection.Exogenous application of GA₃could reduce the total number of nematodes in soybean roots and delay the development of nematodes.In addition,exogenous application of GA₃ could increase the content of endogenous GA₁ and GA₃,up-regulate the expression of mi R159,inhibited the expression of Gm MYB33 gene in soybean and restore the resistance of STTM-mi R159 silencing transgenic soybean hair roots to nematodes.In summary,this study demonstrated that both GA and mi R159 positively regulated the resistance of soybean to cyst nematode,and GA also positively regulated the expression of mi R159,that is,mi R159 involved in the GA-regulated resistance pathway to cyst nematode.