Over-Expression Of GmKR3,a TIR-NBS-LRR Type R Gene,Confers Resistance To Multiple Viruses In Soybean

Soybean mosaic virus(SMV)is found in many soybean production areas,and SMV infection is one of the prevalent viral diseases that can cause 8% to 50% of yield losses annually under natural growth conditions,Therefore breeding virus resistant soybean cultivars may greatly increase yield.SMV is a member of the Potyvirus genus in the Potyviridae family.The genome of SMV is a single-stranded positive-sense RNA,which can translate to 11 proteins,SMV strains vary in different growing regions.Several resistance loci to different strains have been identified in soybean,with three major resistance loci to SMV being identified,i.e.,Rsv1,Rsv3,and Rsv4,In addition to the three major resistance loci,several other genes such as GmAKT2,GmSN1 and GmeEFlA have been shown to involve in resistance to several SMV strains.In plants,resistance(R)genes are involved in pathogen reorganization and innate immune responses activation.Most R proteins have nucleotide-binding sites and leucine-rich repeat(NBS-LRR)domains,and some of the NBS-LRR type R proteins in dicots have a TIR(Toll/Interleukin-1 Receptor)motifs.R genes are involved in plant resistance to diverse pathogens,including fungi,insects,bacteria,nematodes,oomycetes and viruses,and have been proposed to be responsible for gene-for-gene resistance.Indeed,R genes have been shown to interact directly or indirectly with pathogen effectors and enhance plant innate immune response.The plant hormone abscisic acid(ABA)has long been recognized as a key stress hormone that regulates plant responses to abiotic stresses including drought,cold,osmotic and high salinity.Available evidence suggests that ABA also plays an important role in plant basic immunity.In addition,components in ABA signaling have been shown to involve in virus resistance.For example,the type 2C protein phosphatases(PP2Cs)are key regulators of ABA signaling,and PP2C3 a has been shown to contribute to SMV resistance in soybean.GmKR3(Kefeng1 resistance gene 3),a TIR-NBS-LRR type R protein identified by resistance gene analogs(RGAs),shared higher amino acid sequence similarity with the tobacco mosaic virus resistance protein N.Our previous results indicated that the expression level of GmKR3 was higher in Rsmv1,a soybean cultivar resistant to SMV,when compared with that in Ssmv1,a soybean cultivar susceptible to SMV,Then we found that expression levels of GmKR3 increased in all the three SMV-resistant soybean cultivars examined,but remained unchanged or decreased in all the three SMV-susceptible soybean cultivars in response to SMV inoculation.These results suggest that GmKR3 is likely a potential resistance gene to SMV,but experimental evidence is still lacking.When over-expressed in soybean,GmKR3 enhanced the plant's resistance to a broad-spectrum of viruses,including several strains of SMV,their closely related potyvirus bean common mosaic virus(BCMV)and watermelon mosaic virus(WMV),and the secovirus bean pod mottle virus(BPMV).Importantly,over-expression of GmKR3 does not affect plant growth and development,including yield and the qualities of the seeds.HPLC analysis showed that ABA content increased in the 35S:GmKR3 transgenic plants and in response to virus inoculation in both the wild type and the 35S:GmKR3 transgenic plants.Consistent with this observation,We found that the expression levels of some genes including NCED2,NCED4 and AAO3 A were increased after virus inoculation,however,all the genes examined have similar expression levels in both wild-type and 35:GmKR3 transgenic plants and the expression of two ABA metabolism genes was down-regulated in the 35S:GmKR3 transgenic plants.We also found that expression of Gm04.3,an ABA responsive gene,was up-regulated in the 35S:GmKR3 transgenic plants.We thus examined the DEGs for genes homologous to those that have been reported to involve in the regulation of virus resistance in plants.Two alpha-expansin genes,GmEXPA1 and GmEXPA2,were identified.Quantitative RT-PCR results showed that the expression levels of GmEXPA1 and GmEXPA2 were induced by virus inoculation but reduced in the 35S:GmKR3 transgenic plants.To text if GmEXPA1 and GmEXPA2 may function similarly in soybean,we inoculated virus on the upper parts of the leaves,and then examined virus content in the lower parts of the leaves,virus content in the wild type increased sharply after inoculation.Taken together,our results suggest that 35S:GmKR3 enhanced virus resistance in soybean,and which is achieved at least in part via ABA signaling.