Functional Analysis Of Gma-miR1507a In Resistance To Phytophthora Sojae In Soybean

Soybean is one of the main oil crop sources in the world,easily affected by a variety of environmental factors including abiotic stresses such as drought,salt,flooding or biotic stresses by pests and diseases in its growth and development.Phytophthora stem and root rot,caused by Phytophthora sojae,is one of the most destructive diseases of soybean,and the incidence of this disease has been increasing in several soybean-producing areas around the world.The most effective method to reduce damage would be to grow Phytophthora-resistant soybean cultivars.Therefore,the study of molecular of mechanism of interaction between soybean and P.sojae and grow Phytophthora-resistant soybean cultivars are the most urgent way to solve the problem.miRNAs are a class of endogenous and noncoding small molecular RNA with approximately 18-25 nucleotides(nt)in length.miRNA can reduce the protein levels of their targets through transcript cleavage in plant.miRNAs are admitted to play an important role in plant growth and development,adaptation to stresses,including the regulation of host defences.This study aims at demonstrating the function of legumesspecific miRNA,gma-miR1507a in the soybean response to P.sojae.The main results are follows:1.gma-miR1507a negatively regulate the soybean defense against P.sojaeThe mature sequence,secondary structure,members and promoter sequence of gmamiR1507a were extracted in PNRD.We analyzed the occurrence and distribution of putative cis-regulatory elements on the promoter region of gma-miR1507a.The fungal elicitor responsive element Box-W1and other key cis-acting elements were observed in the gmamiR1507a promoter.The gma-miR1 507a abundance after infected with sojae was detected by stem-loop qRT-PCR.The expression level of gma-miR1507a after P.sojae treatment suggest a potential role of gma-miR1507a in soybean defense against P.sojae.The expression pattern of gma-miR1507a was tissue-specific.We successed obtained artificial miR1507a precursors and insert it into the binary vector pCAMBIA2301.Then we transformed it into soybean hair root by Agrobacterium rhizogenes-mediated genetic transformation.The transformed positive hairy roots were isolated based on the histochemical GUS stain.qRT-PCR analysis showed that the expression levels of gmamiR1507a was up-regulated in gma-miR1507a-transformed hairy roots compared with empty vector-transformed hairy roots.Furthermore,the amiR1507a was stably transformed into soybean by Agrobacterium tumefaciens-mediated genetic transformation,and the gmamiR1507-overexpressing soybean showed enhanced susceptibility to P.sojae.2.GmRGA4 is identified as the target gene of gma-miR1507aNine putative targets of gma-miR1507a were predicted using psRNATarget software.To determine which genes are directly targeted by gma-miR1507a,a modified 5’ rapid amplification of cDNA ends(5’-RACE)protocol was performed.Glyma.04g137800(named GmRGA4)was directly cleaved by gma-miR1507a which contains a NB-LRR domain.3.GmRGA4 positively regulate the soybean defense against P.sojaeTo further investigate whether GmRGA4 plays a role in soybean defense against P.sojae,we searched the GmRGA4 promoter by NCBI and predicted putative cis-acting element of GmRGA4.The functional domain of the GmRGA4 revealed that GmRGA4 is a CNL-like protein belong to RGAs family,which specifically target pathogenic effector proteins inside the host cell,termed effector triggered immunity(ETI).GmRGA4 has two transcript GmRGA4.1 and GmRGA4.2.GFP fusion expression vector with GmRGA4.1 and GmRGA4.2 was constructed and transient expression them in Nicotiana benthamiana leaf by Agrobacterium rhizogenes-mediated genetic transformation,showing that GmRGA4.1 and GmRGA4.2 protein is distributed in the nucleus and cytoplasm.Silencing GmRGA4 in soybean hair root by Agrobacterium rhizogenes-mediated genetic transformation.The transformed positive hairy roots were isolated based on GFP fluorescence and qRT-PCR.transgenic roots silencing GmRGA4 exhibited the longer lesion length of P.sojae infection and greater P.sojae biomass accumulation than that of control.The GmRGA4-silencing soybean root hair showed enhanced susceptibility to P.sojae.GmRGA4 positively regulate the soybean defense against P.sojae.Our research showed that gma-miR1507a-GmRGA4 is a regulator in soybean defense to P.sojae which affects the resistance of soybean to Phytophthora root rot,and it might participates in ETI reaction.